Land and Food Systems, Faculty of
DSpace
UBCV
Tzanidis, Anestis Marios
Master of Science - MSc
University of British Columbia
Campylobacter has been recognized as the leading cause of human gastrointestinal diseases with C. jejuni and C. coli being responsible for ~90% and ~10% of human campylobacteriosis, respectively. In this study, we determined the prevalence and characteristics of these two thermotolerant Campylobacter species in retail chicken in metro Vancouver, Canada. Over 200 chicken samples were collected from different districts and tested for Campylobacter contamination using modified Charcoal Cefoperazone Deoxycholate Agar after enrichment in Preston broth. A multiplex polymerase chain reaction (m-PCR) assay was applied to identify C. jejuni and C. coli strains, followed by antimicrobial susceptibility tests using the broth microdilution method. Aminoglycosides, lincosamides, macrolides, quinolones, tetracyclines and phenicols were the major antimicrobial classes used to determine the antimicrobial resistance patterns of the isolates. Finally, we performed multilocus sequence typing to investigate the population structure and their relationship with Canadian human isolates. A total of 34% of the samples were tested positive for C. jejuni (80%) and C. coli (20%) and the majority were resistant to nalidixic acid at 60% and 69%, followed by resistance to ciprofloxacin at 51.1% and 38.4% for C. jejuni and C. coli, respectively. We did not discover any multi-drug resistant Campylobacter belonging to either species. Nevertheless, 17.8% of C. jejuni and 38.5% of C. coli were resistant to ciprofloxacin, nalidixic acid, tetracycline and doxycycline at the same time. The chicken isolates showed significant genetic diversity with 9 C. jejuni sequence types (STs) belonging to six commonly identified clonal complexes (ST-21, ST-42, ST-48, ST-283, ST-343 and ST-353) and 9 C. coli isolates belonging to one clonal complex (ST-828). This study shed light on the levels, population structure and antimicrobial resistance of C. jejuni and C. coli isolated from chicken products in Vancouver, emphasizing the need to implement additional intervention strategies to control this foodborne pathogen.
PREVALENCE AND CHARACTERIZATION OF CAMPYLOBACTER ISOLATED FROM CHICKEN PRODUCTS IN METRO VANCOUVER by Anestis Marios Tzanidis B.Sc., Aristotle University of Thessaloniki, 2006 M.Sc., University of Thessaly, 2008 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (FOOD SCIENCE) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2019 © Anestis Marios Tzanidis, 2019 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: Prevalence and characterization of Campylobacter isolated from chicken products in metro Vancouver submitted by Anestis Marios Tzanidis in partial fulfillment of the requirements for the degree of Master of Science in Food Science Examining Committee: Xiaonan Lu Supervisor Savvas Hatzikiriakos Supervisory Committee Member Supervisory Committee Member Vivien Measday Additional Examiner iii Abstract Campylobacter has been recognized as the leading cause of human gastrointestinal diseases with C. jejuni and C. coli being responsible for ~90% and ~10% of human campylobacteriosis, respectively. In this study, we determined the prevalence and characteristics of these two thermotolerant Campylobacter species in retail chicken in metro Vancouver, Canada. Over 200 chicken samples were collected from different districts and tested for Campylobacter contamination using modified Charcoal Cefoperazone Deoxycholate Agar after enrichment in Preston broth. A multiplex polymerase chain reaction (m-PCR) assay was applied to identify C. jejuni and C. coli strains, followed by antimicrobial susceptibility tests using the broth microdilution method. Aminoglycosides, lincosamides, macrolides, quinolones, tetracyclines and phenicols were the major antimicrobial classes used to determine the antimicrobial resistance patterns of the isolates. Finally, we performed multilocus sequence typing to investigate the population structure and their relationship with Canadian human isolates. A total of 34% of the samples were tested positive for C. jejuni (80%) and C. coli (20%) and the majority were resistant to nalidixic acid at 60% and 69%, followed by resistance to ciprofloxacin at 51.1% and 38.4% for C. jejuni and C. coli, respectively. We did not discover any multi-drug resistant Campylobacter belonging to either species. Nevertheless, 17.8% of C. jejuni and 38.5% of C. coli were resistant to ciprofloxacin, nalidixic acid, tetracycline and doxycycline at the same time. The chicken isolates showed significant genetic diversity with 9 C. jejuni sequence types (STs) belonging to six commonly identified clonal complexes (ST-21, ST-42, ST-48, ST-283, ST-343 and ST-353) and 9 C. coli isolates belonging to one clonal complex (ST-828). This study shed light on the levels, population structure and antimicrobial resistance of C. iv jejuni and C. coli isolated from chicken products in Vancouver, emphasizing the need to implement additional intervention strategies to control this foodborne pathogen. v Lay Summary Bacteria in the genus Campylobacter account for most cases of bacterial gastroenteritis in Canada. Raw poultry meat especially chicken is considered to be the primary cause of human campylobacteriosis. Campylobacter gastroenteritis is a self-limiting disease for most people, but it can pose a high health risk with severe complications to specific groups. Furthermore, the abuse of antibiotics for the treatment of human and animal disease has increased the levels of resistance in Campylobacter to the existing antimicrobials. We used a multiplex polymerase chain reaction to detect C. jejuni and C. coli in retail chicken meat, followed by antimicrobial resistance tests and multilocus sequence typing, a molecular technique that investigates the genetic structure of the bacterial population between the chicken isolates and human isolates in Canada. This thesis provides insight on the prevalence and characteristics of Campylobacter in poultry products in British Columbia. vi Preface The author, Anestis Marios Tzanidis, under the guidance of Dr. Xiaonan Lu, carried out the experimental work, analyzed the data and wrote the thesis. The work is original and has not been previously published. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 1.1 Campylobacter taxonomy ............................................................................................... 1 1.1.1 Characteristics of the family Campylobacteraceae ..................................................... 1 1.1.2 The genus Campylobacter .......................................................................................... 3 1.1.3 Characteristics of Campylobacter genome ................................................................. 4 1.2 Subtyping Campylobacter .............................................................................................. 5 1.3 Human infection.............................................................................................................. 7 1.3.1 Infective dose of Campylobacter ................................................................................ 7 1.3.2 Symptoms of campylobacteriosis ............................................................................... 8 1.3.3 Campylobacter species implicated in human campylobacteriosis .............................. 8 1.4 Epidemiology of Campylobacter .................................................................................. 11 viii 1.4.1 Incidence of campylobacteriosis ............................................................................... 11 1.4.2 Routes of infection .................................................................................................... 11 1.4.3 Seasonality of Campylobacter infections ................................................................. 12 1.4.4 Age, gender and ethnicity ......................................................................................... 13 1.4.5 Outbreaks of campylobacteriosis .............................................................................. 14 1.5 Campylobacter pathogenesis ........................................................................................ 15 1.5.1 Complications associated with campylobacteriosis .................................................. 17 1.5.1.1 Campylobacteriosis associated with Guillain-Barré syndrome and the related Miller-Fisher syndrome .................................................................................................... 18 1.5.1.2 Campylobacteriosis associated with reactive arthritis ...................................... 20 1.5.1.3 Campylobacteriosis associated with irritable bowel syndrome ........................ 20 1.5.2 Estimating the burden of Campylobacter ................................................................. 21 1.6 Campylobacter reservoirs ............................................................................................. 24 1.6.1 Survival of Campylobacter ....................................................................................... 26 1.6.1.1 Survival of Campylobacter in the environment ................................................ 27 1.7 Campylobacteriosis associated with chicken consumption .......................................... 30 1.8 Antimicrobial resistance of Campylobacter ................................................................. 31 1.8.1.1 Resistance to quinolones ................................................................................... 33 1.8.1.2 Resistance to macrolides and lincosamides ...................................................... 34 1.8.1.3 Resistance to aminoglycosides ......................................................................... 35 1.8.1.4 Resistance to tetracyclines ................................................................................ 36 1.8.1.5 Resistance to phenicols ..................................................................................... 37 ix 1.9 Hypotheses and Objectives ........................................................................................... 37 1.9.1 Hypotheses ................................................................................................................ 37 1.9.2 Objectives ................................................................................................................. 38 Chapter 2: Application of a multiplex-PCR assay for the identification of C. jejuni and C. coli in chicken meat products......................................................................................................39 2.1 Introduction ................................................................................................................... 39 2.2 Materials and methods .................................................................................................. 43 2.2.1 Sample collection ...................................................................................................... 43 2.2.1.1 Bacterial isolation ............................................................................................. 44 2.2.1.2 DNA extraction ................................................................................................. 45 2.2.1.3 Speciation using a multiplex-PCR assay .......................................................... 45 2.2.2 Statistical analysis ..................................................................................................... 47 2.3 Results ........................................................................................................................... 47 2.3.1 Prevalence of Campylobacter in chicken meat samples ........................................... 47 2.3.2 Detection of multiple Campylobacter species in individual samples ....................... 50 2.3.3 Seasonality of Campylobacter in chicken meat samples .......................................... 50 2.3.4 Campylobacter prevalence in chicken products with different packaging status ..... 50 2.4 Discussion ..................................................................................................................... 51 Chapter 3: Antimicrobial resistance profiles of C. jejuni and C. coli chicken isolates ..........58 3.1 Introduction ................................................................................................................... 58 3.1.1 The minimum inhibitory concentration .................................................................... 59 3.1.2 Breakpoints ............................................................................................................... 60 x 3.2 Materials and methods .................................................................................................. 60 3.2.1 Antimicrobial solutions ............................................................................................. 60 3.2.2 Campylobacter inoculations ..................................................................................... 62 3.3 Results ........................................................................................................................... 64 3.3.1 Antimicrobial resistance (AMR) profiles of Campylobacter isolates ...................... 64 3.4 Discussion ..................................................................................................................... 68 Chapter 4: The multilocus sequence typing of C. jejuni and C. coli isolated from chicken and associations with antimicrobial resistance and Canadian clinical isolates......................74 4.1 Introduction ................................................................................................................... 74 4.2 Materials and methods .................................................................................................. 76 4.2.1 Campylobacter isolates and preparation of DNA template ...................................... 76 4.2.2 Amplification and nucleotide sequence determination ............................................. 76 4.3 Results ........................................................................................................................... 78 4.3.1 Detection of housekeeping genes in C. jejuni and C. coli isolates ........................... 78 4.3.2 Assignment of allele numbers, STs and clonal complexes ....................................... 80 4.3.3 Relationship between MLST and antimicrobial resistance ...................................... 85 4.4 Discussion ..................................................................................................................... 87 Chapter 5: Conclusion .................................................................................................................93 5.1 Study outcomes ............................................................................................................. 93 5.2 Future directions ........................................................................................................... 94 Bibliography .................................................................................................................................96 xi List of Tables Table 1.1 Percentage of Campylobacter species implicated in pediatric gastroenteritis in Cape Town, South Africa (Lastovica et al. 2000) .................................................................................. 10 Table 2.1 Primers used for multiplex-PCR assays........................................................................ 46 Table 2.2 Positive rates of chicken products with different packaging status .............................. 51 Table 3.1 Dilutions of the antimicrobial agents to produce the stock solutions ........................... 62 Table 3.2 Antimicrobial test ranges, MIC QC range, and breakpoints used for antimicrobial susceptibility testing* .................................................................................................................... 64 Table 3.3 Antimicrobial resistance profiles of C. jejuni and C. coli isolates ............................... 68 Table 4.1 Oligonucleotide primers for MLST of C. jejuni amplification ..................................... 77 Table 4.2 Oligonucleotide primers for MLST of C. coli amplification ........................................ 77 Table 4.3 Allele numbers, sequence types and clonal complexes of C. jejuni isolates* ............... 81 Table 4.4 Allele numbers, sequence types and clonal complexes of C. coli isolates ................... 81 xii List of Figures Figure 2.1 Gel electrophoresis of m-PCR products from the amplification of 26 presumptive Campylobacter colonies. Lane M: molecular weight marker, 100-bp DNA ladder; Lane 1: positive controls for 16S rRNA, C. jejuni and C. coli; Lanes 2-13, 16, 19-26 and 28: C. jejuni; Lanes 15, 17, 18, 27: C. coli; Lane 14: negative control. ............................................................. 48 Figure 2.2 Prevalence of Campylobacter spp. isolated from different chicken products ............. 49 Figure 2.3 Seasonal prevalence of C. jejuni and C. coli in chicken samples ................................ 51 Figure 3.1 Visual MICs of a C. jejuni strain in Mueller-Hinton broth. Antimicrobials tested in rows A – H: ciprofloxacin, erythromycin, clindamycin, tetracycline, chloramphenicol, gentamicin, florfenicol, and nalidixic acid, respectively. The wells showing the MIC values are circled in red. ................................................................................................................................ 65 Figure 3.2 Antimicrobial resistance of C. jejuni and C. coli isolates. ERY, erythromycin; AZI, azithromycin; TET, tetracycline; DOX, doxycycline; GEN, gentamicin; CLI, clindamycin; CIP, ciprofloxacin; NAL, nalidixic acid; CHL, chloramphenicol; FLO, florfenicol. ........................... 66 Figure 4.1 Gel electrophoresis of PCR products for MLST scheme A) C. jejuni, B) C. coli. Lane M: molecular weight marker, 100-bp DNA ladder; Lane 1-7: aspA, uncA, gltA, glyA, pgm, tkt, and glnA, respectively. .................................................................................................................. 79 Figure 4.2 Minimum spanning tree established on the 9 C. jejuni STs identified in this study and human isolate STs from Canada collected from the MLST database. Each circle represents an ST. The size of the circle is positively correlated to the isolate numbers ..................................... 83 Figure 4.3 Minimum spanning tree established on 5 C. coli STs isolated from retail chicken in this study and compared to human C. coli isolates in Canada acquired from the MLST database. xiii Each circle represents an ST. The size of the circle is proportional to the isolate numbers with the same ST. The branch styles represent the numbers of identical alleles between connected STs (black thick solid line, six alleles; black thin solid line, five alleles; gray thick solid line, four alleles; dashed line, three alleles; and dotted line, two alleles or fewer). The color inside the circle stands for clonal complex. Chicken isolates are shown with a thick yellow border of the circle. ............................................................................................................................................. 84 Figure 4.4 Phylogenetic tree of C. jejuni isolates with the relative clonal complex and resistance profile. ........................................................................................................................................... 86 Figure 4.5 Phylogenetic tree of C.coli isolates with the relative clonal complex and resistance profile. ........................................................................................................................................... 86 xiv List of Abbreviations AMAN: acute motor axonal neuropathy AMR: antimicrobial resistance AP-PCR: arbitrary element primers AZI: azithromycin bp: base pair BPW: buffered peptone water CC: clonal complex CDC: Center for Disease Control CFU: colony forming unit CHL: chloramphenicol CIP: ciprofloxacin CIPARS: Canadian Integrated Program for Antimicrobial Resistance Surveillance CLI: clindamycin CLSI: Clinical and Laboratory Standards Institute DALY: disability adjusted life year DNA: deoxyribonucleic acid dNTP: deoxyribonucleotide triphosphate DOX: doxycycline ECDC: European Center for Disease Control ECOFF: epidemiological cut off xv EFSA: European Food Safety Authority ERY: erythromycin EUCAST: European Community of Antimicrobial Susceptibility Testing FDA: Food and Drug Administration FLO: florfenicol GBS: Guillain-Barré syndrome GEN: gentamicin IBS: irritable bowel syndrome mCCDA: modified charcoal deoxycholate agar MDRGI: multidrug-resistant genomic island MH: Mueller-Hinton MIC: minimum inhibitory concentration MLST: multilocus sequence typing MOMP: major outer membrane protein m-PCR: multiplex polymerase chain reaction NAL: nalidixic acid NARMS: National Antimicrobial Resistance Monitoring System’s OD: optical density PCR: polymerase chain reaction PFGE: pulsed field gel electrophoresis QC: quality control QRDR: quinolone resistance-determining region xvi ReA: reactive arthritis RFLP: restriction fragment length polymorphism rRNA: ribosomal ribonucleic acid ST: sequence type TET: tetracycline VBNC: viable but non culturable wgMLST: whole genome multilocus sequence typing WGS: whole genome sequencing xvii Acknowledgements First and foremost, I would like to thank my supervisor Dr. Xiaonan Lu for giving me the unique opportunity to study in his laboratory, for his confidence in my abilities and for his interest in my progress throughout my degree. Secondly, I want to thank my committee members Dr. Savvas Hatzikiriakos and Dr. Pascal Delaquis, for their time commitment and valuable suggestions to my research project. I am very grateful for the financial support from UBC Faculty of Land and Food Systems and MITACS. My special thanks go to Cassendra (Yunxuan) Chen for her role in relieving the huge workload of this past summer, as well as Emma Sproston for her guidance with the writing of this thesis. I would also like to thank all the Lu lab members for their help in so many ways and for the moments that we have shared in these two years. It was an honor to meet and work next to such wonderful people. Finally yet importantly, I would like to express my entire gratitude to my family. First to my parents who have been with me in love and support throughout this journey, I am grateful for both of you and all that you offer. In the end, it came down to Grace and Elizabeth, who xviii sustained me through the long hours, missed weekends and tiredness. Heartfelt thanks to my wife and daughter, they are the true sunshine in my life. xix Dedication This is the Dedication. I dedicate this thesis to my wife, Grace and to our daughter, Elizabeth for their unconditional love and endless support. They are my backbone and the greatest blessing in my life. 1 Chapter 1: Introduction 1.1 Campylobacter taxonomy 1.1.1 Characteristics of the family Campylobacteraceae It took approximately a century from the first observations in 1886 for scientists to identify what Theodor Escherich described as unidentified spiral bacteria in the feces of children suffering from diarrhea and described the disease as “cholera infantum” (Escherich 1886; Park 2002). Campylobacter was first isolated in 1906 in the United Kingdom by John MacFadyean, a veterinary who investigated high abortion rates (as high as 33%) in cattle and sheep (Skirrow et al. 2006). A few years later in 1916, Smith also investigated infectious abortions in cattle and described the pathogen that he isolated as a spirillum. He assumed that he had isolated the same pathogen as MacFadyean and proposed the name Vibrio fetus due to their spiral shape and physiology (Smithet al. 1919). Jones and colleagues (1946) investigated the infection of cattle that had winter dysentery and identified a Vibrio that they named Vibrio jejuni, which caused animals to suffer from watery diarrhea accompanied with a huge decrease in milk yield. The first well-reported human outbreak occurred in Illinois in 1938 affecting 335 inmates who had consumed raw milk by accident. A pathogen resembling V. jejuni was isolated from 13 out of 39 blood samples (Levy et al.1946). In 1947, V. fetus was also isolated from blood samples of three pregnant women who had been admitted to hospital with high fever and resulted in the abortion of the embryos in two of the three cases (Vinzent et al. 1947). However, the presence of Campylobacter bacteria as an enteric pathogen remained undiscovered until the 1970s due to the lack of selective media that could meet its fastidious growth requirements. Sebald and others 2 differentiated this pathogen from the Vibrio genus and renamed it Campylobacter originating from the Greek words used to describe “curved” and “rod” (Sebald et al. 1963). Since the first description of Campylobacter in 1963, there have been several changes in its taxonomy that reflect the advancement of bacterial taxonomic methodologies and the increasing scientific interest and evidence in this microorganism. Initially, the classification of the microscopically observed spiral organisms was based solely on cell morphology and immunological tests due to the inability to cultivate. Sebald and others used the G+C ratio in genomic DNA as well as Hugh and Leifson’s test for fermentative metabolism to show that V. fetus and V. bubulus were different than other Vibrio species and that they proposed another genus to include these taxa (Sebald et al. 1963). Thomson and coauthors demonstrated the phylogenetic diversity of all the existing Campylobacter species by comparing the 16s rRNA sequences that they used to investigate the relationship between these bacteria and concluded that C. pylori had been wrongly classified together with C. jejuni and C. coli as their 16s rRNA sequences differed significantly (Thompson et al. 1988). Vandamme et al. (1992) utilized rRNA-DNA hybridization as a novel tool to study the phylogenetic relationship in combination with other analyses (e.g. whole-cell protein patterns) in a polyphasic approach to investigate the genetic relationships. As a result, the aerotolerant Campylobacter bacteria were placed in the new genus Arcobacter (Vandamme et al. 2014). The only species of environmental origin was placed in the new genus Sulfurospirillum (Schumacher et al. 1992). The current family of Campylobacteraceae now includes the genera Campylobacter, Arcobacter and Sulfurospirillum and is included in the ribosomal RNA superfamily VI (On et al. 2001). 3 Despite the isolation of Campylobacter from a patient’s fecal and blood samples who was admitted to the hospital experiencing diarrhea and high fever, it was not until 1972 that members of the genus Campylobacter were first recognized as the cause of food-borne illness (Dekeyser et al. 1972). Campylobacter bacteria were identified in patients without symptoms of diarrhea as well as in patients reporting diarrhea. C. jejuni and C. coli were isolated in 5.3% of the patients that reported diarrhea and only in 1.6% of the patients without diarrhea (Dekeyser et al. 1972). One of the most important findings of their study was the high susceptibility of C. jejuni to erythromycin and this antibiotic was then used for the therapeutic tests. In all treated cases, erythromycin enhanced the attenuation of the symptoms and the re-testing of the clinical samples revealed the absence of C. jejuni (Butzler et al. 2004a). In 1974, Butzler and coauthors performed antigenic typing of C. jejuni isolates by agglutination and complement fixation tests that showed the close antigenic relationship between isolates from humans and those from poultry, sheep and pigs (Butzler et al. 1974). 1.1.2 The genus Campylobacter The genus Campylobacter includes 26 known species (Kaakoush et al. 2015).These microorganisms are S-shaped spiral rods (except for C. showae, which is a straight rod)and sizes range from 0.2 to 0.9 μm wide and from 0.5 to 5 μm long (Etoh et al. 1993). Campylobacter cells possess a single polar unsheathed flagellum at one or both ends to enable them to be motile. Its motility patterns include movements of rapid jerking and instantaneous reversing. This “corkscrew-like” movement aids in observing the bacterium microscopically (Silva et al. 2011). Motility is an attribute of all Campylobacter species except for C. gracilis, which is considered 4 as non-motile (Skirrow et al. 1977; Siqueira et al. 2003, Silva et al. 2011). Campylobacter spp. are oxidase- and catalase-positive except C. upsaliensis, which can be negative or weakly positive. They are not able to either oxidize or ferment carbohydrates and hence require alternative carbon sources for growth, such as through the reduction of tri-carboxylic acid intermediates and amino acid substrates (Barros-Velazquezet al. 1999; Leachet al. 1997). Probable carbon sources utilized by Campylobacter cells are several amino acids, including L-serine, L-aspartic acid, L-asparagine, and L-glutamic acid (Line et al. 2010). Furthermore, the addition of organic acids to the basal broth was validated to enhance the growth of Campylobacter (Hinton 2006). These microorganisms are unable to grow under atmospheric concentrations of oxygen and are routinely cultivated under microaerophilic conditions with a relatively high concentration of carbon dioxide and low concentration of oxygen (Butzler et al. 2004). An atmosphere containing 5% oxygen, 10% carbon dioxide and 85% nitrogen is typically used for the incubation of Campylobacter (Stern et al. 1984). In addition, the presence of hydrogen has been identified to enhance the growth of emerging Campylobacter spp. (Lastovica et al. 2006). 1.1.3 Characteristics of Campylobacter genome C. jejuni and C. coli have a single circular DNA molecule with a size between 1.6 and 1.7 megabases and a low GC ratio of approximately 30% (Taylor et al. 1992). The first C. jejuni strain whose genome was sequenced was NCTC11168 and it was discovered that the organization of genes into operons or clusters was absent except for those that control the production of ribosomal proteins and surface modifications (Parkhill et al. 2000). There was also 5 a surprising lack of repetitive DNA sequences with only four repeated sequences identified. Dispersed repeated sequences have been recognized as a potential source of genetic variation and regulation (McClintock 1956). Furthermore, the genome sequencing of different Campylobacter strains revealed the presence of hypervariable sequences, where areas of DNA showed variation at a single base pair between otherwise identical clones and the degree of variation differed between sites. In contrast, the genome of C. coli shows less genomic variation between different strains and is characterized as rather homogeneous (On et al. 2000; Duim et al. 1999). 1.2 Subtyping Campylobacter Different molecular typing methods are used worldwide to give genetic information of infectious pathogens including Campylobacter. Molecular typing can be used to identify strains, their frequency and distribution patterns and to relate them to clinical cases, geographic regions, different hosts, sources or temporal scales. Such molecular typing aids in our understanding of disease, pathogenicity, epidemiology, identifying, and investigating the source of outbreaks. Different methods have different drawbacks that may include inadequate discrimination, poor reproducibility and transferability within and between laboratories and also the inability to assess the genetic relationship between isolates in a quantitative manner (Maiden et al. 1998). Restriction fragment length polymorphism (RFLP) was the first method that was developed for DNA profiling (Li et al. 2009). RFLP uses restriction enzymes to digest the DNA and produce different fragment lengths that are identified using gel electrophoresis. It is also able to identify length variations, which could be a consequence of deletion, restriction or insertion in the DNA (Saghrouni et al. 2013). After the development of PCR, RFLP became outdated as it took weeks 6 to acquire the results and it used a large amount of DNA. In comparison, PCR used very small amount of DNA and the results were produced within a few hours (Tabit 2016). Pulsed-field gel electrophoresis (PFGE) or PCR with repetitive or arbitrary element primers (AP-PCR) have also been used to segregate bacterial strains and they both rely on comparisons of DNA fragments on agarose gels. These methods can identify genetic variation, which accumulates rapidly to a high degree. This can offer a high discriminative power in epidemiological investigations in the short term, but does not provide answers to the epidemiological investigations in a long term or in different geographical regions (Enright et al. 1999). For example, a typing method that has detected no variations within all tested Campylobacter isolates could not be useful to investigate outbreaks caused by this strain or to compare epidemics of the pathogen in different countries. Thus, such a method lacks reproducibility and it is now considered outdated for genotyping (Araujo et al. 2018). Multilocus enzyme electrophoresis (MLEE) uses the genetic variability of selected regions (by restriction enzymes or primers) of the genome and by identifying a small number of alleles (variant forms of genes) it offers a high discriminatory power (Maiden et al. 1998; Enright et al. 1999; Wood 1995). A group of researchers later developed multilocus sequence typing (MLST) scheme to study the populations of Neisseria meningitides, a human pathogen that causes life-threatening disease (Maiden et al. 1998). MLST is based on the same principles as MLEE, but the loci from relatively stable housekeeping genes are selected by specific primers and sequenced. Since then, MLST has become a valuable tool in the study of the population structure and evolution of a large number of bacteria. For each housekeeping gene, different sequences present within a bacterial species are assigned as distinct alleles and, for each isolate, the alleles at each of the 7 loci define the allelic profile or sequence type (ST). The relatedness of STs is described by clonal complexes. Clonal complexes have been described as the clusters of bacterial strains that are genetically closely related (Selander et al. 1987; Maiden et al. 1998). 1.3 Human infection 1.3.1 Infective dose of Campylobacter In a study conducted in Maryland, Black and others revealed that some volunteers (5 out of 10) developed symptoms of campylobacteriosis by ingesting only 800 colony forming units (CFU). The researchers also discovered that the colonization rate increased when they increased the infective dose, but the development of illness did not increase (Black et al. 1988). In fact, the model developed by Black and others showed that the risk of developing illness decreased as the infective dose increased. In a later study, researchers used the data from two outbreaks involving primary school children becoming ill with campylobacteriosis after consuming raw unpasteurized milk and they produced an updated dose-response relationship for Campylobacter infection (Teunis et al. 2005). This newer model shows that the development of disease increased exponentially at lower doses, but then stopped increasing only to reach to the maximum ~100% response when the highest dose was ingested. The above findings suggest that not all people exposed/infected by an intestinal microorganism even at a high dose will become ill and develop symptoms of disease and that they can remain asymptomatic until the pathogen is excreted (Teunis et al. 2005). In another study, scientists discovered that the infective dose of C. jejuni required for the development of illness was as low as 360 CFU (Hara-Kudo et al. 2011). An intermediate dose of 8 9 x 104 CFU/mL was shown to inflict the highest ratio of illness according to a mathematical model developed by Medema and colleagues (Medema et al. 1996). These studies, in combination with the findings in another study of Rose et al. (1991) that the number of Campylobacter cells needed to cause human illness are much lower than other pathogens such as Salmonella, Shigella or Vibrio, probably explains why Campylobacter is the most commonly occurring entero-pathogen in the developed world (Medema et al. 1996; Rose et al. 1991). 1.3.2 Symptoms of campylobacteriosis Campylobacter infection normally has an incubation period of between one and three days, but it can take as long as ten days for the first symptom to appear (Koenraad et al.1997). Symptomatic Campylobacter infections are most commonly expressed with fever, severe abdominal cramps, headaches and diarrhea often containing blood (Ternhag et al. 2007; Helms et al. 2005). These infections are typically sporadic and usually self-limiting and do not require treatment(Silva et al. 2011). A patient with campylobacteriosis could carry and excrete Campylobacter cells for up to three weeks post infection. Campylobacter cell counts in the clinical samples from the infected patients ranged between 106and 108 CFUg-1(Taylor et al. 1993). 1.3.3 Campylobacter species implicated in human campylobacteriosis Several studies on human isolates conclude that C. jejuni accounts for 80-90% of Campylobacter infections and C. coli for 10% approximately in the developed world (Gillespie et al. 2002; Devane et al. 2005). Lawson and colleagues conducted a survey on 3,378 human isolates and discovered that except for the most prevalent species (89% C. jejuni and 18% C. 9 coli) other Campylobacter species were present at a much lower rate. C. upsaliensis, C. hyointestinalis and C. lari accounted for 2%, 0.6% and 0.2%, respectively (Lawson et al. 1999). Interestingly, data from the African continent appear to be quite different. In a ten-years study conducted at the Red Cross Children’s Hospital in South Africa, Lastovica and colleagues examined stool samples from 19,535 children that had been admitted to the hospital with diarrhea (Lastovica et al. 2000). Table 1.1 summarizes various Campylobacter species isolated in this study and the percentage of illnesses that they caused. In another study, Engberg and others suggested that C. concisus should be considered as a normal commensal of human gut, thus justifying the findings of the South African study. They also emphasized the difficulty in screening human diarrheal samples for Campylobacter species other than C. jejuni, C. coli and C. lari as the methods routinely used by laboratories cannot isolate all taxa successfully (Engberg et al. 2000). Ten years later, Zhang and colleagues investigated the presence of C. concisus isolated from saliva samples of healthy individuals and patients with a chronic gastrointestinal disease. The age groups ranged between 3 and 60 years and the results showed a high prevalence for both healthy individuals and patients (97% and 100% respectively) with no statistical differences between the healthy and the group experiencing symptoms of disease as well as between different genders. The interesting finding in this study was that the rates of C. concisus were significantly different between the age group 3-5 years and older children or adults. Zhang and coauthors also concluded that specific strains of C. concisus should be considered as commensals of the human oral cavity and assumed that the different rates between the very young and the older children and adults could be associated with the larger subgingival 10 space of adult teeth, which would provide larger cavities that would enhance the growth of C. concisus (Zhang et al. 2010). Table 1.1 Percentage of Campylobacter species implicated in pediatric gastroenteritis in Cape Town, South Africa (Lastovica et al. 2000) Campylobacter species % Positive C. jejuni subsp. jejuni 31.4 C. concisus 23.6 C. upsaliensis 23 C. jejuni subsp.doylei 9.2 C. coli 2.9 C. hyointestinalis 1.3 C. fetus 0.17 C. curvus 0.1 C. rectus 0.1 C. sputorum 0.1 In a case-control study of entero-pathogens associated with childhood diarrhea, it was discovered that the isolation rate from children with symptoms of Campylobacter-mediated disease in developing countries was not significantly different from that of the asymptomatic cases. Researchers hypothesized that more frequent contact with animals, manure and raw products in the rural areas led to a higher level of exposure to the zoonotic pathogens (Albert et al. 1999; de Wit et al. 2001). On the other hand, the isolation rates from asymptomatic people in the developed countries (e.g. Netherlands) were as low as 0.5% (de Wit et al. 2001). 11 1.4 Epidemiology of Campylobacter 1.4.1 Incidence of campylobacteriosis Globally, Campylobacter is estimated to cause 166 million illnesses per year (Percivalle et al. 2016) but there is a wide variation between different regions. In the areas where foodborne disease surveillance programs are well established, the annual incidence of campylobacteriosis often appears to be higher. Oceania ranks first with 112–154 cases per 100,000 in the population, followed by Europe (59.8 per 100,000) and 14.3 per 100,000 in North America (Kaakoush et al. 2015). In comparison, during 2015 there were 23 cases per 100,000 people in Canada (PHAC, 2017). It is estimated that 4 million cases of domestic foodborne illnesses occur in Canada each year (Thomas et al. 2015). Campylobacter infections account for 8.42% of the domestic foodborne illnesses (approx. 145,000 cases ) and rank Campylobacter 3rd after norovirus and Clostridium perfringens (Thomas et al. 2015). 1.4.2 Routes of infection The majority of Campylobacter infections are sporadic with the handling or consumption of raw or undercooked poultry meat being the primary vehicle for Campylobacter infection (Skirrow 1977). However, other factors have been identified as statistically significant to cause Campylobacter mediated disease. These factors include travel, consumption of raw milk or undercooked barbecued meat, consumption of contaminated drinking water or activities associated with recreational waters, consumption of contaminated vegetables, transmission from pets as well as contact with wild and domestic animals or their feces (Montgomery et al. 2017; Adak et al. 1995; Dasti et al. 2010; Verhoeff-Bakkenes et al. 2011; Friedman et al. 2000). 12 In a study conducted by Gillespie and others, it was proposed that risk factors should be identified to the species level. Researchers noted that C. coli infections had been mostly reported by people who had previously consumed bottled water or eaten specific types of meat (Halal meat, pâté, offal, meat pies) in the weeks prior to the development of illness. On the other hand, C. jejuni infections were reported by people who had been in contact with animals. These differences between the species are thought to be significant when trying to identify transmission routes for Campylobacter (Gillespie et al. 2002). 1.4.3 Seasonality of Campylobacter infections A comparison of seasonal trends in a UK-based study that monitored the incidence of campylobacteriosis cases for nearly a decade showed a strong seasonal pattern (Louis et al. 2005). Factors such as daily average temperature, precipitation, and sunshine were significantly associated with campylobacteriosis rates. In another study, Nylen and colleagues compared the seasonal trends among10 countries and discovered that the seasonal patterns of human cases increased during distinct weeks within the warmer seasons of the year (Nylen et al. 2002). In a more recent study conducted in Scotland, several factors were proposed to play a significant role in the incidence of campylobacteriosis. The higher temperature in the environment was not the sole source of seasonality. Increased foreign and domestic travel during the warmer months was an important factor as well. Another element justifying Campylobacter seasonality was assumed to be the turning of farm animals to pasture along with humans spending more time outdoors in the rural areas (e.g. hiking, camping) emphasizing the fecal-oral route of infection. Furthermore, 13 the increased activity of both wildlife and humans initiated by the mild weather was believed to play a significant role (Strachan et al. 2013). 1.4.4 Age, gender and ethnicity In a UK cohort study, demographic data from a population-based surveillance scheme for Campylobacter infections in England and Wales were compared to study the relationship between age, gender and the incidence of campylobacteriosis(Gillespie et al. 2019). An increased incidence in the group of less than 2-yearold was observed and probably due to infants and young children being more likely to visit primary-care physicians. In addition, there was a peak in the incidence of people aged 17 years that showed a significantly higher incidence in males than females. Another recent study showed that hormonal changes during puberty might be related to the successful growth of Campylobacter cells present in the human gut (Cogan et al. 2007). Furthermore, the incidence of Campylobacter infections between 22 and 69 years appeared to remain stable and decreased after the age of 70 years (Gillespie et al. 2019). Geissler and others had also reported similar findings in a study on the American population where infants until the age of 4 years showed the highest rates of campylobacteriosis (Geissler et al. 2017). Interestingly, in the same study where a comparison among different racial/ethnic groups was attempted, it was discovered that the incidence of Campylobacter infections was higher for American Indians and Alaskan Natives compared to the Caucasians. These findings were in contrast with the data from New Zealand where people of European descent were reported to have the highest rate (>90%) of campylobacteriosis and Pacific residents had a much lower incidence of ~6.4% (Eberhart-Phillips et al. 1997). 14 1.4.5 Outbreaks of campylobacteriosis Campylobacter outbreaks have most commonly been associated with the consumption of poultry meat, unpasteurized milk or untreated water (Rosenfield et al. 1985; Pearson et al. 2000; Heuvelink et al. 2009; Clark et al. 2003). In a study conducted in Australia, Rosenfield and others confirmed the first outbreak of Campylobacter enteritis to be reported in the country. The outbreak involved 7 of 17 people consuming a chicken casserole dish at a restaurant and the onset of symptoms varied from 1 to 5 days. Campylobacter jejuni Penner serotype was isolated from the patients’ clinical samples as well as from three raw fresh chicken samples closely associated with the implicated chicken. Serotyping revealed that the isolates of both sources were identical (Rosenfield et al. 1985). In a more recent Australian outbreak in 2012, 15 out of 57 guests who attended a birthday party developed symptoms of campylobacteriosis 1 to 5 days after the consumption of chicken liver pâté. Eight patients sought medical care, one person was hospitalized and fecal samples were provided by three patients in total. Laboratory results confirmed the presence of C. jejuni in both human clinical isolates and chicken liver samples (McPherson et al. 2012). Chicken liver pâté has been implicated with numerous outbreaks worldwide (Little et al. 2010; Glashower et al. 2017; Scott et al. 2015). In a Scottish outbreak study, O’Leary and colleagues identified the first continuous Campylobacter outbreak in Scotland that affected 48 people in a 3-week period. All the patients that developed symptoms of campylobacteriosis had dined at the same restaurant and had consumed chicken liver pâté. Although Campylobacter was not isolated from the chicken livers due to a change in the method of cooking the pâté, the 15 infection rate of the customers who had consumed the pâté prior to the change was calculated to be 91% (O’Leary et al. 2019). In 2006, Whyte discovered that cooking chicken liver at an internal temperature of 70-80°C for 2-3 min is efficient in inactivating all naturally occurring Campylobacter bacteria (Whyte et al. 2006). According to the US Center for Disease Control, Campylobacter outbreaks are not commonly reported, considering how often people get sick from this pathogen, but the frequency has been increasing. The average number of outbreaks reported each year from 2004 to2009 was 28. In comparison, it was 59 from 2010 to2012 and 35 from 2013 to 2015 (CDC 2018). Between 1992 and 2009, 143 outbreaks were reported in England and Wales, United Kingdom (Little et al. 2010). Surveillance data reporting outbreaks are severely lacking for many developing as well as developed nations. 1.5 Campylobacter pathogenesis Most Campylobacter spp. preferably colonize the mucus layer of the gastrointestinal tract of poultry and mammals. A model of infection was proposed by Konkel and coauthors who studied the virulence attributes of C. jejuni in a piglet model due to their anatomical similarity with humans. Furthermore, piglets are considered as a good model since they develop clinical symptoms such as bloody diarrhea, histopathological lesions and epithelial cell degeneration, similar to humans suffering from campylobacteriosis (Konkel et al. 2001). Motility, chemotaxis, adherence and toxin production seem to play a major role in the virulence. It has been proposed that C. jejuni primarily colonizes the jejunum and ileum as well as the colon of humans followed by migration towards the mucus-filled enterocrypts of the small intestine via the paracellular or 16 transcellular route. Konkel and others suggested that Campylobacter cells synthesize novel proteins that promote the interaction with the host cells once in the small intestine. Moreover, they release toxins that appear to destroy the tips of the villi, resulting in villous atrophy (Konkel et al. 2001). The role of flagella is not only limited to aid in the adherence of the cells to the host receptors but also to assist in the movement of the cells across the epithelium. Adhesins are proteins that facilitate the adherence and establishment of the pathogenic cells on the host intestinal cells. The levels of adhesins synthesized by the invading pathogens correlate with the degree to which C. jejuni cells adhere to the cultured host cells (Fauchere et al. 1986). In a previous study, Konkel and colleagues discovered that CadF was an adhesin found in all the C. jejuni and C. coli chicken isolates they tested and it is required for Campylobacter cells to bind on the fibronectin on the host cells (Konkel et al. 1999). Once bound on the host cell, bacteria invade it with the secretion of specific proteins. In another study, invasion antigens (Cia) were validated to play a significant role in the internalization of the pathogenic bacterial cell into the host cell. The absence of Cia protein secretion resulted in a significant reduction in internalization (Konkel et al. 1999). Following internalization, bacteria have to survive under the stress of reactive oxygen species, such as hydrogen peroxide, found in the host cell. To neutralize toxic oxygen species, bacteria utilize superoxide dismutase and catalase encoded by the kata gene. A C. jejuni katA mutant could not be recovered from the J774A cell line (Konkel et al. 2001). Iron plays an important role in bacterial growth. In the host, free iron is not available so that most bacteria have developed mechanisms to scavenge it. Furthermore, in the host, iron is 17 found in complexes with transferrin and lactoferrin and is not readily available. Outside the host cell, bacterial cells frequently utilize siderophore-mediated iron uptake. Outer membrane receptors bind the iron loaded siderophores and transport them into the cell. The cfrA gene in C. jejuni encodes a protein that is believed to be the siderophore receptor (Konkel et al. 2001). In a Danish study, researchers investigated the presence of seven putative virulence and toxin genes in C. jejuni and C. coli isolated from pig and cattle farms. Bang and co-authors discovered a high prevalence (>80%) of the cytolethal distending toxin gene cluster in the isolates. Furthermore, they noticed a high prevalence of genes associated with adhesion (cadF), motility and adhesion (flaA), iron acquisition (ceuE) and toxin production (cdtB) (Bang et al. 2003). 1.5.1 Complications associated with campylobacteriosis Campylobacter infections are usually self-limiting and do not require medical treatment. However, Campylobacter cells can spread from the gastrointestinal tract to other organs causing pancreatitis, peritonitis, cholecystitis and intestinal hemorrhage (Acheson et al. 2001). More rarely, Campylobacter-induced bacteraemia has led to meningitis, endocarditis, septic arthritis and neonatal sepsis. These rare cases (<1%) mainly involve the very young, very old or immunocompromised patients (Acheson et al. 2001). 18 1.5.1.1 Campylobacteriosis associated with Guillain-Barré syndrome and the related Miller-Fisher syndrome Complications of campylobacteriosis can lead to Guillain-Barré syndrome (GBS) with a median incidence of 1.3 cases per 100,000 population as well as the related Miller-Fisher syndrome (1-2/1,000,000) (Gupta et al. 2016; Hughes et al. 1997). These autoimmune disorders can paralyze the nerves and eventually lead to fatal respiratory paralysis in ~5% of GBS cases (CDC 2017; Nachamkin et al. 1998; Salloway et al. 1996). It might take one to three weeks, or in some cases up to 6 months, for the appearance of the first paralytic symptoms due to post Campylobacter GBS (Nachamkin et al. 1998; Poropatich et al. 2010). Antecedent infection, surgery or prior trauma are the major factors contributing to the development of GBS (Yang et al. 2016). Among the subset of patients with GBS whose symptoms are triggered by an infection, 20–50% are attributed to an initial C. jejuni infection (Nyati et al. 2013). In 1995, there were 65 admissions of campylobacteriosis in New Zealand and eight of the patients (>12%) developed GBS. This rate is considerably higher comparing to the general rate of 1.3 cases per 100,000. An implementation of stricter hygiene measures on poultry meat in the following year resulted in decreased rates of campylobacteriosis and subsequent decreased rates of GBS (Withington et al. 1997). According to Hadden and others, ascending paralysis initially occurs at the patient’s legs and as the disease progresses it moves to the upper body and cranial nerves (Hadden et al. 2001). In 2000, the C. jejuni genome was sequenced and three sets of genes, namely cst-II, cgtA, and cgtB, were identified to be involved in the biosynthesis of sialic acid (Parkhill et al. 2000). Sialic acid is a component of Campylobacter cell outer membrane that mimics the structure of specific 19 human gangliosides located on the peripheral nerves. Initially, GBS was regarded as a demyelinating disease of the peripheral nerves, but the axonal form was later considered to be the primary form. Axonal GBS is often characterized by electrophysiological and pathological evidence of axonal degeneration of the motor nerves together with functional conduction failure (Hiraga et al. 2005). It is also characterized by acute paralysis and loss of reflexes without sensory loss. Anti-GD1a antibodies (anti-ganglioside antibodies) are associated with the axonal (acute motor axonal neuropathy [AMAN] associated with degeneration of motor nerves) form of GBS, whereas anti-GM1 antibodies are seen in both AMAN and demyelinating (acute inflammatory demyelinating polyneuropathy) forms (Nachamkin et al. 2002). Lipopolysaccharides of C. jejuni strains isolated from patients with axonal forms of GBS were determined to share glycoconjugate epitopes with the peripheral nerve. The same epitopes including GM1-, GQ1b- and GD1a-like moieties as well as anti-GM1 and anti-GD1a antibodies have been identified in some of the patients with axonal GBS (Oomes et al. 1995; Cogan et al. 2007; Sheikh et al. 1998). This molecular mimicry is thought to play an important role in the evasion of host’s immune system and the development of autoimmune disease (Endtz et al. 2000). Specific C. jejuni clonal complexes have been associated with an increased GBS incidence. Nielsen and coauthors used MLST to characterize 122 clinical samples from humans including 18 from patients with confirmed GBS. They identified that 64% of the isolates were plotted in 3 clonal complexes namely, ST-21, ST-22 and ST-45. The GBS isolates were significantly grouped in the ST-22 clonal complex, which was consistent with the isolates that were registered on the PubMLST database (Nielsen et al. 2010) 20 1.5.1.2 Campylobacteriosis associated with reactive arthritis Reactive arthritis (ReA) follows approximately 1-3% of the reported campylobacteriosis cases and has also been associated with Chlamydia, Yersinia, Salmonella and Shigella (Altekruse et al. 1999; Colmegna et al. 2004). ReA has been described as an inflammation of the synovial membrane of a joint that typically appears within 4 weeks after an intestinal or urogenital infection. Colmegna and others discovered the presence of bacterial antigens in the joint, which are believed to be synthesized in the joint or elsewhere in the body (Colmegna et al. 2004). The symptoms vary from mild arthralgia to severely disabling polyarthritis and have a predilection for the knees and ankles of the patient, but can also be present in small joints or as tenosynovitis (Pope et al. 2007). The treatment of ReA with antibiotics has not been shown to have any effect on the expression of the condition as it is not clear if antibiotic treatment of the primary infection can inhibit the subsequent development of ReA. Furthermore, it is not known if the use of antibiotics can shorten the ReA course and if a prolonged use of antibiotics is required to ensure intracellular eradication of the pathogens. Early suppression of the pathogenic infection may diminish the antigen load and modulate the severity of ReA expression (Kingsley et al. 1996; Leirisalo-Repo 1998). Therefore, ReA is thought to be an immune-mediated disease (Colmegna et al. 2004). 1.5.1.3 Campylobacteriosis associated with irritable bowel syndrome Following a Campylobacter infection, some patients have been diagnosed with irritable bowel syndrome (IBS), which is a chronic medical condition associated with abdominal pain, 21 bloating and altered bowel habits, such as constipation and diarrhea (Gwee 2010). Post-infectious IBS can appear in a large percentage of patients infected by several enteric pathogens including Campylobacter, Salmonella and Shigella (Spilleret al.2000). One of the highest ever reported rates of post-infectious IBS (~36%) was after a campylobacteriosis outbreak in the town of Walkerton, Ontario where citizens were infected after drinking the contaminated tap water (Marshall et al. 2006; Clark et al. 2003). Previous studies had characterized IBS as a psychosomatic illness attributing it mainly to the increased stress levels of the patients (Toneret al. 1990).A bio-psychological model linking the psychosomatic concept of the disease with the sequela of severe gastrointestinal infections was proposed (Gwee 2010). It has been found that stress can aggravate IBS symptoms and can be associated with psychological co-morbidities which are seen between 42% and 61% of patients with medical disorders. In addition, IBS patients are also likely to indicate lower pain thresholds and also exhibit higher levels of enterochromaffin cells in the gut mucosa, which contain serotonin (a neurotransmitter) (Tanaka et al. 2011). Other studies have also indicated that the brain-gut axis is a bidirectional and integrated system in which psychological factors (thoughts, feelings, memories etc.) can lead to neurotransmitter release that affects sensory, motor, immune and inflammatory functions (Tanaka et al. 2011). 1.5.2 Estimating the burden of Campylobacter Several attempts have been made to estimate the burden of foodborne illness at a national level. The large variability of factors that plays a significant role in estimating the burden (e.g., environmental factors, pathogens, hosts, and under-diagnosis) makes it challenging to fully 22 understand the burden caused by the foodborne pathogens (Thomas et al. 2013; Smith 1919; Scallan et al. 2011; Scallan, Griffin, et al. 2011; Havelaar et al. 2012). In order for a sick person to be recorded by the surveillance system in Canada, the following must occur: (1) the patient seeks care, (2) the patient has a sample (stool, urine, or blood) requested, (3) the patient submits a sample for testing, (4) the sample must be tested with a test capable of identifying the causative agent, and finally (5) this positive test result must be inputted into the surveillance system (Thomas et al. 2011). A rough estimation of ~11 million foodborne illness cases were generated by the Public Health Agency of Canada based on the values acquired from the estimates in United States (Mead et al. 1999). For the U.S.-CDC (Center for Disease Control) and Canada, norovirus, C. perfringens, Campylobacter and non-typhoidal Salmonella account for almost 90% of the pathogen-specific burden. However, the overall estimate is slightly higher for the U.S.-CDC than the Canadian estimate. Approximately one in six Americans contracts a foodborne illness annually, compared to that of one in eight Canadians (Thomas et al. 2013). In contrast to the previously reported ~11 million foodborne illness cases in Canada, Thomas and colleagues estimated that there are ~4 million cases of domestically acquired foodborne illnesses annually, of which 1.6 million cases are related to 30 specified pathogens. The rest of the cases (~2.4 million) are attributed to unspecified agents. Among those accountable for the largest numbers of illnesses are: norovirus (1 million), C. perfringens (177,000), Campylobacter spp. (145,000), and non-typhoidal Salmonella (88,000). These findings are significantly different from the initial Canadian estimate of 11 million cases, which had used values from the former estimates in U.S. to apply to the Canadian population in total. This newer approach by Thomas and colleagues is more consistent with the U.S.-CDC 23 estimates as it uses a more specific case definition for acute gastrointestinal illness in their survey questionnaire and has also been adopted by the Canadian Notifiable Disease Surveillance System (Thomas et al. 2013; Government of Canada, 2012). To quantify the health impact of Campylobacter, the disability adjusted life year (DALY) metric model was adopted and it is currently the most popular summary measure of population health (Murray et al. 2013; Devleesschauwer et al. 2017). The DALY is a key measure in the global burden of disease studies as it summarizes the occurrence and impact of morbidity and mortality, with one DALY representing the loss of one year in full health. DALYs are calculated by adding the number of years of life lost due to premature mortality and the number of years lived with disability, adjusted for severity. Lackner and colleagues estimated 967 DALYs for acute Campylobacter-mediated gastroenteritis in Germany, of which 98% were associated with morbidity and 2% with mortality (Lackner et al. 2019). Other recent estimates of the burden of Campylobacter range from 22,500 DALYs in the USA, 18,222 in Australia, 3,633 in Netherlands, and 1,568 in New Zealand (Skarp et al.2016). Currently, there is no data available for Canada. As far as the economic burden of campylobacteriosis is concerned, the USDA ranks Campylobacter infections as fifth among 15 pathogens. Campylobacteriosis is believed to impose an economic burden of $1.9 billion per year in the country from which $1.1 billion is attributed to GBS. This is further translated that less than 0.25% of the infections are responsible for over 50% of the economic burden. The rest is distributed in 34% ($658 million) due to resulting deaths from acute illness and only ~10% ($202 million) due to non-hospitalized and 24 hospitalized cases of campylobacteriosis (Hoffmann et al. 2015). There is no available official estimation of the economic burden of Campylobacter in Canada at the moment. 1.6 Campylobacter reservoirs Campylobacter exist as harmless commensals of several warm-blooded wild and domestic animals, especially birds where Campylobacter is carried in the intestines asymptomatically and shed in their feces (Kaakoush et al. 2015). Stanley and others showed that cattle and sheep are potential Campylobacter reservoirs as contamination occurs in several levels in the dairy farm. Young calves had the higher rates of Campylobacter colonization and stored and dispersed slurries of cattle provided a reservoir for scavenging birds and insects to transmit the disease further (Stanley et al.2003). In another study, Boes and colleagues investigated the prevalence of Campylobacter in pigs at different levels, including slaughterhouse and farm, with or without the presence of cattle and sheep. No significant differences were identified in the prevalence between the different herds at the slaughterhouse level. However, pigs were identified to have a higher prevalence (98%) of C. coli compared to the other types of farm animals, such as cattle (6.8%) and poultry (3.8%) (Boes et al. 2005). Campylobacter is strongly associated with avian hosts since this microbe grows optimally at 42°C, near the internal body temperature of poultry (41°C) and other birds (Horrocks et al. 2009). Poultry can be colonized by Campylobacter through horizontal transmission from other poultry, other animals and environmental sources as well as from fecal contamination external to rearing facilities (Newell et al. 2003). Generally, Campylobacter spp. colonize the caecum and colon of poultry in high concentrations. They can also be identified in the alimentary tract of birds due to the coprophagic nature of chickens and shared drinking water, which facilitates 25 rapid transmission of Campylobacter in broiler flocks (Tsubokura et al. 1997). Shanker and co-authors showed that it took between 3-7 days for a whole flock (~90%) of chickens to be colonized by Campylobacter from the initial inoculation in their feeds (Shanker et al.1990). Campylobacter has also been frequently detected in the feces of numerous other animals including pigs, rodents, cattle, and wild birds (Aarestrup et al. 1997; French et al. 2009; Meerburg 2007). The genetic diversity of C. jejuni isolated from different farm animals was investigated in a UK-based survey in 2003. Colles and colleagues identified a total of 30 genotypes and MLST revealed that all isolates were grouped in one of the nine C. jejuni clonal complexes previously identified from humans. They also discovered evidence for the association of certain clonal complexes with particular types of animals. Bacterial isolates grouped in the ST-45 complex were predominantly isolated from poultry and were totally absent among sheep and accordingly isolates ST-61 and ST-42 predominantly isolated from sheep were not found in poultry. Interestingly, C. jejuni isolates belonging to ST-21 complex were distributed among different isolation sources. Comparison of the isolates of animal origin with isolates from human clinical samples showed that six of the clonal complexes were found in both groups (Colles et al. 2003). Campylobacter has also been isolated from environmental water sources, such as rivers, streams, lakes and coastal waters (Guy et al. 2018; Gu et al. 2013; Obiri-Danso et al.2000). During the investigation regarding a Campylobacter outbreak in Walkerton, Ontario, scientists were able to trace back the source of contamination to a particular C. jejuni stain. After a heavy rain, bacteria from cattle manure from a nearby farm were able to enter the ground water and as a result infected those who consumed water from that well (Clark et al. 2003). Since 30oC is the 26 minimum temperature required for the growth of this microbe and it is sensitive to atmospheric oxygen levels, Lee and colleagues concluded that Campylobacter survives rather than grows in the aquatic environment (Lee et al. 1998). 1.6.1 Survival of Campylobacter Although they are thermophilic and grow best at 42°C, Campylobacters can be easily inactivated at a cooking temperature of 60°C for 15 minutes or 57.5°C for 30 minutes (Barros-Velazquezet al. 1999; Stintzi 2009). The growth of Campylobacter in food matrices is rare since they are sensitive to oxidative stress and they cannot grow at temperatures below 30°C (Park 2002). Refrigeration and freezing are techniques used to inhibit the growth of microorganisms in different agri-food commodities. Although Campylobacter cells cannot grow below 30°C (Lee et al. 1998), physiological activities such as oxygen consumption, catalase activity, ATP generation, and protein synthesis still occur at as low as 4°C. Furthermore, Hazeleger and coauthors observed that chemotaxis and aerotaxis were also present at all the tested temperatures (4°C-46°C), indicating that Campylobacter cells were able to move to more favorable locations (Hazeleger et al. 1998). Lee and coauthors also studied the effect of cold stress on the survival and growth of C. jejuni cells that had been artificially inoculated onto chicken skin. C. jejuni 81116 was able to survive during long term storage at -70°C and -20°C in different packaging atmospheres, such as carbon dioxide, microaerophilic, nitrogen, vacuum, and normal atmospheres (Lee et al.1998). In another study, Bhaduri and others investigated the survival of three C. jejuni strains artificially inoculated into ground chicken and onto chicken skin, followed by storage at both 4°C and -20°C. Campylobacter populations were reduced by 0.56 to 1.57 log10 27 CFUg-1 in ground chicken and by 1.38 to 3.39 log10 CFUg-1 on chicken skin after two weeks of storage at -20°C. Much lower reductions were observed during refrigeration for 3-7 days, specifically by 0.34 to 0.81 log10 CFU/g in ground chicken and by 0.31 to 0.63 log10 CFU/g on chicken skin (Bhaduri et al. 2004). These findings highlight the fact that despite not being able to grow outside a host, Campylobacter cells can survive for long periods especially at cool temperatures. On the other hand, their survival rate declines more at temperatures between 25 and 30oC (Smith et al. 2016). Thus, the role that food compositions (e.g., protein, fat, ion content, salt, acid, viscosity, etc.) play in bacterial survival highlights the fact that both refrigeration and freezing cannot fully substitute for the safe handling and appropriate cooking of poultry meat (Bhaduri et al. 2004; Speck et al. 1977). Campylobacter bacteria are also sensitive to nutritional and pH stresses (Klančnik et al. 2009). Ideally, they require a pH between 6 and 8 and are unable to multiply below pH 4.9 (Park 2002). In addition, a concentration of sodium chloride higher than 2% as well as dry surfaces deter their growth (Doyle et al. 1982a; Doyle et al. 1982b). To visualize Campylobacter susceptibility to air-drying, Kusumaningrum and others inoculated dry stainless-steel surface with 105and 107 CFU/100 cm2of C. jejuni. After 4 h of air drying at room temperature, no C. jejuni could be recovered (Kusumaningrum et al. 2003). 1.6.1.1 Survival of Campylobacter in the environment Current scientific evidence suggests that when Campylobacter is encountered in the environment it is commonly associated with fecal contamination (Dearlove et al. 2016). In a study aiming to determine the survival time of Campylobacter in animal feces, Ahmed and 28 others compared the survival times of naturally colonized broiler flocks with artificially inoculated feces of Campylobacter-free flocks. The samples were incubated at 20±1oC and 40-60% RH (relative humidity) and the cultivability of C. jejuni was investigated at the same time of the day for 10 consecutive days. The authors discovered that the survival of the same strain of C. jejuni that had been artificially inoculated in poultry feces ranged from 72 to 96 h whereas for the naturally colonized flocks it survived from 120 to 144 h (~6 days). These results demonstrate that Campylobacter cells can survive outside the host for many days post excretion. It also reveals the importance of contaminated feces as a reservoir for Campylobacter in the environment (Ahmed et al. 2013). In another study, Inglis and colleagues studied the survival of Campylobacter in cattle feces that had been treated with a tetracycline versus a non-treated control group. Campylobacters could be readily isolated from pen manure for up to 15 days in the control group and for 126 days in the group that was treated with tetracycline. Furthermore, they tested for C. jejuni DNA quantities during the 10-month composting period of the cattle manure. They used centrifugation in combination with ethidium monoazide (EMA) to significantly reduce (>90%) the amplification of C. jejuni DNA that did not originate from cells with intact outer membranes. Interestingly, there were no significant differences between the two samples (EMA and non-EMA treated). These results indicate that Campylobacter can persist in cattle manure for extended periods of time (Inglis et al. 2010). French and others studied the population structure of C. jejuni associated with cattle farmlands. They collected and examined feces from livestock and wild animal as well as samples of environmental water and soil within a 100 km2 area from the farms. They identified that in their majority, cattle isolates were grouped in the ST-61 complex, whereas isolates from wildlife 29 and water were more likely to belong to the ST-45 complex. They also discovered several uncommon sequence types (STs). Their findings show the natural transmission pathways and the variable potential of different C. jejuni strains to survive in different environments (French et al. 2005). Bacteria can adjust to the changing environmental conditions utilizing their two-component system (TCS) that serves as a basic stimulus-response mostly through the differential expression of the targeted genes (Stock et al. 2000; Mascher et al. 2006). Two proteins, a sensor histidine kinase (HK) and a response regulator (RR), are the major components of most bacterial TCS. The role of HK is to sense certain signals in the extracellular environment and therefore activate the kinase by the autophosphorylation of a conserved histidine residue. Consequently, the phosphoryl group is transferred to an aspartate residue of the RR by histidine phosphorotransferesase, leading to either stimulation or repression of the target gene expressions (Capra et al. 2012). Parkhill and colleagues reported the existence of nine putative response regulators, six putative HKs and five TCSs in the genome of C. jejuni NCTC11168 (Parkhill et al. 2000). Another mechanism that appears to play a significant role in the survival of Campylobacter in extreme environmental conditions is their ability to form biofilms. Biofilm is an extracellular polymeric substance typically made of extracellular polysaccharides, proteins, lipids and encased bacterial cells (López et al. 2010; Reuter et al. 2010). Biofilm formation is a bacterial response to various factors, such as cellular recognition to a specific attachment site onto a solid substrate surface, nutritional limitation or the presence of antimicrobials in the agro-ecosystem (An et al. 2007). Campylobacter can form biofilms in the aquatic environment, on glass or even stainless 30 steel (Guntheret al. 2009). A few studies have identified that bacterial cells enter the VBNC state when forming biofilms and therefore require less nutrients. Furthermore, a prolonged stationary phase enhances bacterial survival by offering additional resistance to aerobic stress, peroxide sanitizers and heat (Buswell et al. 1998; Murphy et al. 2006; Alter et al. 2006). 1.7 Campylobacteriosis associated with chicken consumption According to the European Food Safety Authority, broiler meat may account for 20% to 30% of the ~9 million cases of human campylobacteriosis in the European Union annually, while 50% to 80% may be attributed to the chicken reservoir as a whole (broilers as well as laying hens) (EFSA 2011). However, many infections and outbreaks have also been traced back to raw milk, contaminated water and contact with pets and other farm animals (Corry et al. 2001; Burakoff et al. 2018; Montgomery et al. 2017; Pedati et al. 2019). Consumption of chicken meat was identified as the major risk factor in two case-control studies conducted in New Zealand. In fact, 80% of the hospitalized patients were reported to have consumed chicken meat in the previous days (Eberhart-Phillips et al. 1997; Ikram et al. 1994). In a Norwegian case-control study the chicken meat consumed at the home level was also identified to be one of the risk factors associated with the sporadic cases of Campylobacter infections (Kapperud et al. 1992). Rosenquist and others attempted to assess the risk factors of the annually increasing numbers of human campylobacteriosis in Denmark. They estimated that the Danish population consumes ~201 million chicken servings per year that led to ~14,000 people getting infected by C. jejuni and C. coli. Cross-contamination through cutting boards is also thought to be a risk 31 factor. However, it was identified that the incidence decreased when the boards were washed with warm water and soap between food preparations (Rosenquist et al. 2003).Another study highlighting the major role that poultry meat plays in Campylobacter infections was conducted in Belgium following the dioxin crisis in chicken meat attributed to the contamination of feed components. Researchers noted that the withdrawal of all poultry products from the Belgian market resulted in a significant reduction of 40% of the cases of human campylobacteriosis (Vellinga et al. 2002). 1.8 Antimicrobial resistance of Campylobacter Resistance to antimicrobial agents can be categorized as either intrinsic or acquired resistance. Intrinsic resistance is an inherent characteristic and is the result of vertical transmission of genes to progeny. All C. jejuni and C. coli isolates are considered to be intrinsically resistant to most of the cephalosporins, rimfapicin, trimethoprim, sulfamethoxazole, vancomycin, penicillin, novobiocin and bacitracin. On the other hand, acquired resistance can be developed by a chromosomal mutation or acquisition of foreign DNA, such as plasmid and transposon (Taylor et al. 1988). The main mechanisms involved in antimicrobial resistance include decreased wall permeability to the antimicrobial compounds, an efflux pump system across the cell membrane, expression of alternative proteins, camouflaging of the target, mutation of the antimicrobial target site as well as enzymatic degradation or alteration of the antimicrobial compounds (Whitehouse et al. 2018). Despite Campylobacter enteritis being a self-limiting disease that does not usually require antimicrobial therapy, in some cases prolonged or relapsing illness may occur and antibiotic 32 treatment may be necessary, especially in children, elderly and immunocompromised people (Blaser et al.2014). Campylobacter infections have also been known to cause abortions and stillbirths in pregnant women, thus, making it essential to administer antibiotic treatment (Allos et al. 2010). In these cases, macrolides, primarily erythromycin (or one of the newer macrolides azithromycin or clarithromycin) are the drugs of choice for diagnosed cases of Campylobacter (Whitehouse et al. 2018). Fluoroquinolones (e.g., ciprofloxacin) are also commonly used since they are the drugs of choice for unverified diarrheal diseases, such as traveler’s diarrhea (Aarestrup et al. 2001). The levels of C. jejuni resistance to erythromycin have remained relatively stable and low (<10%) in most parts of the world, except Thailand and Sweden that showed a significant increase between 1980 and 1997. Compared to C. jejuni, the reported rate of erythromycin resistance in C. coli is significantly higher in the majority of reporting countries. Certain developing countries have reported higher rates of erythromycin resistance (>50% for C. jejuni and >90% for C. coli) than those in the developed world, which could be due to different local patterns in the use of this antibiotic to treat animals and humans (Allos et al. 2010). The 2013 CDC report estimates that Campylobacter bacteria resistant to the aforementioned antibiotics maycause~310,000 illnesses and 28 deaths per year in the United States. Similar data for Canada is currently unavailable. However, in a study conducted in Alberta it was determined that the levels of Campylobacter resistance to erythromycin were very low and that they have remained stable for the last 20 years. In the EU, levels of ciprofloxacin ranged from moderately low in some countries to extremely high in some others with a total of 13% resistance to this antimicrobial. Data for erythromycin resistance in the European countries are currently unavailable or under-reported (CDC 2013; ECDC 2018; PHAC 2016; Gibreel et al. 2004). 33 1.8.1.1 Resistance to quinolones DNA gyrase is an enzyme (in the Type II topoisomerases subclass) encoded by gyrA and gyrB genes and can introduce negative supercoils into DNA. In the form of a tetramer (two A and two B subunits), gyrase binds to DNA while the subunits wrap the DNA into a positive supercoil. The enzyme catalyzes the supercoiling reaction aided by the binding of a molecule of ATP. When ATP is not present, gyrase removes the negative supercoils from DNA (Drlica et al.1997).The role of gyrase is particularly important in chromosome function as the maintenance of negative supercoiling activates the chromosome for all processes involving strand separation. Quinolones (e.g., ciprofloxacin and nalidixic acid) can inhibit DNA synthesis and result in bacterial death by binding to DNA gyrase using the energy obtained from the hydrolysis of ATP, to relax positive supercoils and allowing DNA and RNA polymerases to move (Sutormin et al. 2019). Campylobacter resistance to quinolones has been attributed to mutations within the gyrA gene at the quinolone resistance-determining region (QRDR). These gene mutations result in a single amino acid substitution in the QRDR of the GyrA protein that results in Campylobacter resistance to quinolone antimicrobials. This is in contrast to other Gram-negative bacteria where double mutations are required (Luo et al. 2005). An operon of three genes that includes cmeA, cmeB, and cmeC, encodes a periplasmic fusion protein, an inner membrane drug transporter and an outer membrane protein, respectively, and function together as a multidrug efflux pump where its role is to remove a wide range of drugs from the cell cytoplasm (Lin et al. 2002; Ge et al. 2005). CmeABC is believed to assist in the emergence of gyrA mutants that otherwise would be easily inhibited by even low doses of 34 quinolones. In addition, mutations within the chromosomally-encoded CmeABC multidrug efflux pump are also involved in Campylobacter resistance to quinolones. The major mechanism of resistance to quinolones in Campylobacter is via point mutations in gyrA. Additionally, CmeG, a new putative efflux pump was described and noted to confer resistance to structurally unrelated antibiotics as well as antioxidants (Iovine 2013a). 1.8.1.2 Resistance to macrolides and lincosamides Macrolides and lincosamides are separate antimicrobial classes with different structures that inhibit protein synthesis by binding to the ribosome, including the 23S rRNA and ribosomal proteins L4 and L22 (Whitehouse et al. 2018). However, they share overlapping binding sites in the peptidyl transferase region in domain V of the 23S rRNA gene at positions 2074 and 2075. The resistance mechanisms include a) mutations in 23S rRNA genes and ribosomal proteins L4 and L22, b) methylation through the erm gene and modification of the target site, c) CmeABC multidrug efflux pump and reduced membrane permeability, and are described in more details below (Bolinger et al. 2017). These three types of Campylobacter resistance to macrolides (e.g.erythromycin and azithromycin) and consequently lincosamides(e.g. clindamycin) existed within Campylobacter or Gram-negative bacteria. Mutations occurring at these positions alter the binding site of these antimicrobials and disrupt the attachment of these to the bacterial ribosome (Gibreel et al. 2006). Mutations within the 23S rRNA gene have been identified in three copies of the targeted gene as the mechanism conferring resistance to macrolides and lincosamides, but only two are needed at a time (Gibreel et al. 2005). 35 A study conducted by Qin and others reported for the first time the presence of a modification of the targeted bacterial ribosome by methylation of the erm(B) gene in Campylobacter. The erm(B) gene encodes a ribosomal methylase and is located in a chromosomal multidrug-resistant genomic island (MDRGI) to provide resistance to both macrolides and lincosamides. The MDRGI is believed to have originated from Gram-positive bacteria via horizontal transmission to C. jejuni and C. coli via natural transformation (Qin et al. 2014). A few studies have revealed the role of CmeABC efflux pump in both the intrinsic and acquired resistance of Campylobacter to erythromycin (Lin et al. 2002; Mamelli et al. 2005). When the CmeABC efflux pump was inactivated, Campylobacter cells regained their susceptibility and showed intermediate to low levels of resistance to erythromycin (Mamelli et al. 2005). 1.8.1.3 Resistance to aminoglycosides Aminoglycosides (e.g., gentamicin) are antimicrobial compounds that inhibit the synthesis of proteins through binding to the prokaryotic ribosomes. According to Ramirez and others, the most prevalent mechanisms of aminoglycoside resistance in both Gram-negative and Gram-positive bacteria are through enzymatic modifications to inactivate the antimicrobials (Ramirez et al. 2010). The enzymes that can modify aminoglycosides can be divided into three classes depending on the reactions that they catalyze, namely N-acetyltransferases, O-adenyltransferases and O-phosphotransferases. Each class of the aminoglycoside-modifying enzymes has a unique resistance profile that is based on the type of enzymatic modification and site of modification 36 (Zhao et al. 2015). The researchers identified the existence of an aminoglycoside 2ʺ -phosphotransferase resistance gene (aph(2ʺ)), which is believed to encode the most important enzyme associated with a high level of gentamicin resistance in enterococci (Zhao et al. 2015). 1.8.1.4 Resistance to tetracyclines Tetracyclines (e.g. tetracycline and doxycycline) are a class of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site (Chopra et al.2001). The basic mechanisms that are responsible for the resistance to tetracyclines include the CmeABC efflux pump, enzymatic inactivation of antimicrobial compounds, and protection of ribosomal binding site of tetracycline by ribosomal protection proteins (RPPs), including Tet(O) and Tet(M) (Alfredson et al. 2007). For Campylobacter the RPPs are encoded by the tet(O) gene appears to be the main resistance mechanism (Iovine 2013b). The tet(O) gene has been identified to confer tetracycline resistance to levels as high as 512μg/mL (Gibreel et al. 2004). Connel and co-authors identified that Tet(O) confers resistance by blocking the binding of tetracycline from its primary binding site on the ribosome and allows protein synthesis to continue (Connell et al. 2003). The pTet plasmid is a multidrug resistant plasmid commonly identified in C. jejuni and C. coli that carries the tet(O) gene as well as multiple aminoglycoside resistant genes, such as cpp2, cpp3 and cpp4. According to its GC content, sequence homology, and codons sequences the pTet plasmid is believed to have originated from Gram-positive bacteria (Chen et al. 2013). 37 1.8.1.5 Resistance to phenicols In prokaryotes, non-fluorinated (chloramphenicol) or fluorinated (florfenicol) phenicols are highly effective inhibitors of protein synthesis. Their mechanism of action focuses on the prevention of peptide chain elongation by binding to the 50S ribosomal subunit of 70 ribosomes (Schlünzen et al. 2001). Phenicols are used against a wide spectrum of Gram-positive and Gram-negative bacteria. The most common mechanism of resistance to chloramphenicol (but not to florfenicol) is from chloramphenicol acetyltransferases (CAT), encoded by a plasmid carrying the cat gene (Wieczorek et al. 2013; Wang et al. 1990). Resistance to phenicols in Campylobacter is also conferred by target site mutations or modifications within the 23S rRNA by Cfr(C) rRNA methyltransferase or enhanced extrusion by efflux pumps (Shen et al. 2018). Campylobacter cells are not intrinsically resistant to phenicols and generally the prevalence of phenicols resistance identified in Campylobacter isolates is relatively low. However, a recent study from the United States identified a gene similar to the cfr gene, which was detected in 10% of C. coli strains isolated from five dairy farms (Tang et al. 2017). 1.9 Hypotheses and Objectives 1.9.1 Hypotheses Hypothesis 1: We hypothesize that both C. jejuni and C. coli are prevalent in chicken meat products sold in metro Vancouver. 38 Hypothesis 2: We hypothesize that clonal complexes will be discovered that contain both chicken and human Campylobacter isolates. Hypothesis 3: We hypothesize that the antimicrobial tests on the selective Campylobacter isolates will show the presence of multi-drug resistant profiles. 1.9.2 Objectives Objective 1: To investigate the prevalence of C .jejuni and C. coli isolated from retail chicken meat in metro-Vancouver. Objective 2: To apply MLST to investigate the population structure of Campylobacter isolated from retail chicken and its relationship to human isolates in the MLST database. Objective 3: To determine the antimicrobial resistance patterns of the isolated Campylobacter to a wide range of commonly used antibiotic classes. 39 Chapter 2: Application of a multiplex-PCR assay for the identification of C. jejuni and C. coli in chicken meat products 2.1 Introduction Development of a method that is reliable to recover and rapidly identify Campylobacter spp. from environmental samples is still challenging. Difficulties commonly encountered when trying to detect Campylobacter spp. from a variety of sources, including the low numbers present in/on the sample matrices, the state of the cells present (e.g. injured, viable but non-culturable cells [VBNC]) as well as the presence of natural inhibitors. Enrichment is necessary to aid in the recovery of injured bacterial cells as well as to offer conditions that enable cells that are present in low numbers to multiply up to a certain concentration that can be detected (Doyle et al. 1982). A large number of different enrichment broths and agars have been formulated in the last few decades to enhance the recovery of Campylobacter that may be in a injured or stressed state or in low numbers, such as in environmental and food samples (Corry et al. 2001). Commercial media are often supplemented with oxygen quenchers, such as blood or charcoal. Antimicrobial compounds are also supplemented to inhibit the growth of other competitive microflora to which Campylobacter cells are intrinsically resistant. These antimicrobials maybe present in the purchased media or added from either supplement kits or laboratory prepared antibiotics at the required concentrations. The antimicrobials facilitate easier identification of Campylobacter that need a relatively long incubation period by preventing unwanted overgrowth by faster growing, potentially competing background microflora (Griffiths et al. 1990). Several comparative studies have shown that the employment of different isolation methods using different types of media, 40 supplements or different incubation times have produced different results of determining the prevalence of Campylobacter spp. within a single matrix and can influence the overall prevalence of certain strains (Baserisalehi et al. 2004; Tangvatcharin et al. 2005; Jorgensen et al. 2002). For example, in a UK study, 3 selective broths for Campylobacter showed different isolation rates where Exeter broth was significantly more effective in isolating C. jejuni from the environmental samples than both Preston and Bolton broth that yielded the same isolation rate. However, for chicken fecal, soil or litter samples there were no significant difference in the isolation rates of C. jejuni and C. coli between the 3 different broths (Williams et al. 2012). Routinely, enrichment is then followed by conventional agar plating to obtain a single colony for each isolate prior to identification tests. The purification of individual colonies is necessary to ensure that a single strain has been isolated and is achieved by at least two consecutive subcultures of 48 h. For phenotypic identification, Campylobacter isolates undergo several different types of biochemical tests, where the pattern in the array of results (positive or negative) is used to identify the species. However, differentiation to species can be inaccurate and limited. For example, Linton and others showed that ~5% of C. jejuni isolates were misidentified as C. coli because they did not express hippurase, an enzyme that breaks down hippurate (Linton et al. 1997). Waino and colleagues identified that phenotypic methods of speciation should ideally be supplemented with genotypic methods, such as PCR. Employment of such genotypic methods reduces the time and cost, is more accurate, and allows simultaneous identification of multiple species (Wainø et al. 2003). PCR amplifies DNA to enable the detection of specific and targeted genomic sequences. Typically, PCR consists of a series of 20-40 repeated temperature changes (thermal cycles) with 41 the temperatures used in every step being dependent on the enzyme used for DNA synthesis (polymerase), the concentrations of bivalent ions and dNTPs as well as the melting temperatures (Tm) of the primers (Rychlik et al. 1990). A PCR protocol includes the following steps: 1) initialization, where the heating of the reaction chamber is achieved at a temperature of 94-96oC for 1-10 min; 2) denaturation: heating of the reaction chamber at 94-96oC for 20-30 sec that causes breakage of the hydrogen bonds between complementary bases yielding two single-stranded DNA molecules; 3) annealing: the reaction temperature is lowered to 50-65oC for 20-40 sec to allow annealing of the primers to the DNA template (the determination of the right annealing temperature is critical as it affects efficiency and specificity); 4) extension/elongation: the temperature of this step depends on the DNA polymerase used. Commonly, a temperature of 75oC is used and during this step, DNA polymerase synthesizes new DNA strands utilizing the free dNTPs from the reaction mixture. 5) The final hold: this steps cools the reaction chamber to 4-15oC for an indefinite time and may be employed for a short term storage of the PCR products (Rychlik et al. 1990). Due to its high sensitivity, specificity, speed, simplicity and low cost of the assay, PCR is used widely in a variety of biological disciplines and applications, which include clinical diagnosis, discovery of genetic disease risk, as well as identification of toxin genes, resistance genes, virulence genes, and speciation genes and can also be used for DNA fingerprinting in forensic medicine, agriculture and archaeology (Garibyan et al. 2013; Newton et al. 1997). In the previous works, discrimination between C. jejuni and C. coli by PCR required two or three PCR reactions for each single DNA extraction and the equivalent number of agarose gel electrophoresis analyses (Linton et al. 1997). Chamberlain and colleagues were the first to 42 describe a multiplex-PCR (m-PCR) assay that was used to detect deletions in the dystrophin gene that causes a common genetic disease in humans (Chamberlain et al. 1988). The method was described as a traditional PCR assay, but multiple primer sets were used within a single PCR reaction to produce the amplicons of varying sizes. According to the authors, annealing temperatures needed to be optimized and similar to all the primers’ sets and the sizes of the targeted amplicons were different enough to be discriminated on a standard agarose gel. The m-PCR method described by another group was able to identify Campylobacter to the species level but 3 (one C. curvus, one C. helveticus and one C. hyoilei) out of the 70 reference strains from 24 species of Campylobacter, Arcobacter and Helicobacter, produced a same size band as C. coli and subsequently gave a false-positive result. Therefore, this method was not reliable enough to be used in the routine speciation of C. jejuni and C. coli (Harmon et al. 1997).The m-PCR requires further considerations in the design and running than a single PCR reaction. Different targets in the reaction can compete with each other for limited reaction resources (dNTPs), resulting in the detection of templates that are more abundant. An amplification bias also exists in the m-PCR amplification efficiency and can be related to the nucleotide sequence of primer and target primer site, the AT/GC ratio along with other primer binding factors and reagent concentrations and proportions in the m-PCR master mix (MgCl2, dNTPs) (Elnifro et al. 2000). For the purpose of this objective, we utilized a m-PCR assay previously designed (Denis et al. 1999). The same protocol was previously used by the EU Reference Laboratory for the speciation of C. jejuni and C. coli (EURL-AR 2013). 43 2.2 Materials and methods All media and reagents were purchased from Sigma-Aldrich (Oakville, ON, Canada) unless indicated differently. Blood Free Preston Broth was used as the selective broth and supplemented with 4 antimicrobials, namely rimfapicin (10 mg/L), polymyxin B (7 mg/L), trimethoprim (10 mg/L), and cycloheximide (10 mg/L). Modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) supplemented with the four antimicrobials used in Preston broth at the same concentrations along with cefoperazone (at 128 mg/L) and amphotericin B (at 4 mg/L) was used as the selective agar. Mueller-Hinton Agar (BD Difco, FisherScientific, Toronto, ON, Canada) supplemented with 5% defibrinated sheep blood was used as non-selective agar for culturing and isolating single Campylobacter colonies. 2.2.1 Sample collection Fresh chicken meat products were purchased from a variety of retail stores in metro-Vancouver (BC, Canada) between January and November 2019. These sampling sites belong to densely inhabited and busy areas of the city. Dependent on the availability of fresh meat retail stores in the area, 15 to 30samples were collected each month for a total of 11 months. In total, 206 chicken samples were purchased that included whole chicken wings and middle joints (n = 83), whole chicken legs or thighs (n = 64), whole or half chicken breasts (n = 41), chicken necks and backs (n = 8), chicken livers (n = 5) and chicken gizzards (n = 5). The availability of the last three types of chicken was low. Although this may be considered as a limiting factor, their inclusion in this study gives a more representative picture of the prevalence of C. jejuni and C. coli on more types of fresh chicken and identifies the potential risk of consumers being infected 44 with campylobacteriosis from the ingestion or mishandling of these products. Lastly, we included both pre-packaged and bulk chicken meat to investigate the difference in the prevalence of Campylobacter in regards of the packaging status of the poultry. The chicken samples were transported to the laboratory on ice within 2 hours and kept at 4oC until processed within 24 hours of purchase. 2.2.1.1 Bacterial isolation Each chicken sample was placed in a sterile bag (VWR, Mississauga, ON, Canada) and buffered peptone water (BPW) was then added at a ratio of 1mL per 2.5 g of sample. The bag containing the sample and BPW was massaged thoroughly for 1-2 minutes to allow bacterial extraction. For the enrichment analysis, we used the protocol described by the International Organization for Standardization in ISO 10272-1 as previously modified by Ma and others where1 mL of the chicken rinse solution was added to 9 mL of supplemented blood-free Preston broth (Sigma-Aldrich, Oakville, ON, Canada) (Ma et al. 2017). The sample was pre-enriched at 37oC for 4 hours under microaerophilic conditions (10% CO2) with shaking (175 rpm). The mixtures were then incubated for 48 h under the same conditions at 42oC. Ten microliters of the enrichment broth were streaked onto mCCDA and incubated for another 48 hours under the same microaerophilic conditions at 42oC. Single presumptive Campylobacter colonies that exhibited the typical morphology (grey, silver, wet or dry, flat or slightly raised) were individually collected from each mCCDA plate and sub-cultivated onto Mueller-Hinton agar (BD, Franklin Lakes, NJ) supplemented with 5% sterile defibrinated sheep blood. The number of colonies picked from every mCCDA plate for 45 further identification varied between 2 and 5 per sample. All the Campylobacter isolates were stored at -80oC in vials containing 40% glycerol, 45% Mueller-Hinton broth and 5% sterile defibrinated sheep blood for future MLST and antimicrobial tests. 2.2.1.2 DNA extraction Two to three medium-sized colonies were picked with a tip of a pipette and suspended in 100 μL of sterile distilled water in a 1-mLEppendorf tube. Subsequently, the tubes were heated to100°C for 10 min, followed by cool down on ice and centrifugation for 2 min at 15,000 rpm. The supernatant was then collected and placed in a clean Eppendorf tube and used as the DNA template. 2.2.1.3 Speciation using a multiplex-PCR assay A multiplex-Polymerase Chain Reaction (m-PCR) assay as previously described by Denis and others was used for simultaneous detection of the Campylobacter genus and the two Campylobacter species, namely C. jejuni and C. coli(Denis et al. 1999). Three sets of primers were purchased from Thermo Fisher Scientific (Toronto, ON, Canada) and the sequences are shown in Table 2.1. The 16S rRNA binding site is specific to the genus Campylobacter, the mapA gene is specific to C. jejuni and the ceuE gene is specific to C. coli. 46 Table 2.1 Primers used for multiplex-PCR assays Binding sites Sequences (5´ → 3´)a Amplicon size (bp) 16S rRNA F: ATCTAATGGCTTAACCATTAAAC 857 R: GGACGGTAACTAGTTTAGTATT mapA F: CTATTTTATTTTTGAGTGCTTGTG 589 R: CTTTATTTGCCATTTGTTTTATTA ceuE F: AATTGAAAATTGCTCCAACTATG 462 R: TGATTTTATTATTTGTAGCAGCG a F, forward; R, reverse The m-PCR mixture (20μL total in volume) was prepared in a 0.2-mL PCR tube containing 10 μL of 2x TaqFroggaMix Plus (FroggaBio, Toronto, ON, Canada),2 μL of sterile DD water, 2 μL of DNA template, 0.5 μM 16S rRNA primers, 0.5 μM ceuE primers and 1 μM mapA primers. DNA amplification was carried out using a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA) using the following PCR program: initial denaturation step at 95°C for 10 min, followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 55.6°C for 30 sec and extension at 72°C for 30 sec, with a final extension step of 72°C for 10 min. The PCR products were stained using1% (v/v) SYBR safe™ DNA gel stain, followed by gel electrophoresis using a 1.5% agarose gel ran at 120V for 40 min after which the gel was visualized using a Bio-Rad ChemiDoc MP gel imaging system (Bio-Rad, Hercules, CA). During the optimization experiments, we observed that all negative controls had primer-dimer formation (Figure 2.1). It has been suggested that the formation of primer-dimers could be used as an internal control for PCR amplification (Rasmussen et al. 1996). The absence or primer-dimers could suggest that there was either an inhibition problem in the sample or a 47 reagent failure. Subsequently, the primer-dimer formation reduces the likelihood of reporting a false negative result. 2.2.2 Statistical analysis We compared the prevalence of Campylobacter in different chicken products and with different packaging status using chi-squared test in Microsoft Excel. A value of P< 0.05 was considered statistically significant. 2.3 Results 2.3.1 Prevalence of Campylobacter in chicken meat samples A total of 73Campylobacter isolates from 70 positive chicken samples were speciated using this m-PCR method. Each Campylobacter species produced two amplicons: one for the genus at a size of 857 bp (16S rRNA) and one for the species either at a size of 589 bp (mapA) for C. jejuni or 462 bp (ceuE) for C. coli (Figure 2.1). A total of 70 (34.0%) of chicken samples were positive for Campylobacter. C. jejuni was the predominant species being detected in 57 of the 70 samples (78.1%) compared with 16 (21.9%) samples positive for C. coli. 48 Figure 2.1 Gel electrophoresis of m-PCR products from the amplification of 26 presumptive Campylobacter colonies. Lane M: molecular weight marker, 100-bp DNA ladder; Lane 1: positive controls for 16S rRNA, C. jejuni and C. coli; Lanes 2-13, 16, 19-26 and 28: C. jejuni; Lanes 15, 17, 18, 27: C. coli; Lane 14: negative control. The prevalence rates of Campylobacter on different chicken parts were 31.7% for breasts (n = 41), 37.5% for legs and thighs (n = 64), 21.7% for wings (whole and middle joints) (n = 83), 100% for fresh livers (n = 5), 40% for gizzards (n = 5), and 87.5% for necks and backs (attached necks and backs were treated as a single sample) (n = 8). Using the chi-squared test, the prevalence of Campylobacter in different chicken parts was not significantly different (P> 0.05). Figure 2.2 shows the prevalence of C. jejuni and C. coli in different chicken parts. 49 Figure 2.2 Prevalence of Campylobacter spp. isolated from different chicken products *3 chicken thighs carried both C. jejuni and C. coli 31.7 33.321.7 205027.807.81.21004037.57.831.722.91006087.534020406080100120Breastsn = 41Legs + thighsn = 64Wingsn = 83Liversn = 5Gizzardsn = 5Necks & Backsn = 8Totaln = 206Campylobacterprevalence (%)C. jejuniC. coliTotal034.4* 50 2.3.2 Detection of multiple Campylobacter species in individual samples Three chicken thighs tested positive for both C. jejuni and C. coli. Two of those samples came from the same batch and the third sample was collected from a different retailer in downtown Vancouver. 2.3.3 Seasonality of Campylobacter in chicken meat samples Spring showed the highest overall peak of Campylobacter prevalence in chicken at 91.1% of which 80.0% was C. jejuni and 20%C. coli, respectively. The prevalence of C. jejuni in the chicken meat samples was at its highest in spring, then appeared declined and remained stable in the summer and autumn quarters had a much lower prevalence over the colder winter months (Dec-Feb) (Figure 2.3). On the other hand, the prevalence of C. coli followed an increasing trend from December through August with no C. coli being detected during the fall. 2.3.4 Campylobacter prevalence in chicken products with different packaging status We tested 46 pre-packaged and 160 bulk (without packaging) chicken samples. Table 2.2 shows the contamination rates in regards of the different packaging status. Using the chi-squared test we determined a significant difference in the Campylobacter contamination rate between pre-packaged and bulk chicken meat (P< 0.05). 51 Table 2.2 Positive rates of chicken products with different packaging status Packaging status No. of samples No. of positive samples No. (%) of positive samples Pre-packaged 46 20 43.5 (28.3 + 8)* Bulk (unpackaged) 160 50 31.3 (27.6 + 8)* * 1 packed and 2 bulk samples carried both C. jejuni and C. coli Figure 2.3 Seasonal prevalence of C. jejuni and C. coli in chicken samples 2.4 Discussion In our study, 206 retail chicken parts were collected from metro-Vancouver outlets for the isolation and characterization of Campylobacter. A m-PCR assay was used to identify Campylobacter isolates to the genus and species level (C. jejuni and C. coli) 6.58028.6 28.63.711.125010.291.153.628.60102030405060708090100Dec-Feb Mar-May Jun-Aug Sep-NovCampylobacterprevalence (%)C. jejuniC. coliTotal 52 simultaneously. All C. jejuni isolates produced two DNA fragments of 857 bp and 589 bp and all C. coli isolates produced two DNA fragments of 857 bp and 462 bp (Figure 2.1). No other target or non-specific amplicons of other molecular sizes were observed. As shown in Figure 2.2, 34% of the chicken samples (70 of the 206) were contaminated by Campylobacter with C. jejuni and C. coli at 27% and 7%, respectively. To date, there is very little data available on the prevalence of C. jejuni and C. coli on poultry at the retail level in the province of British Columbia. Previous studies conducted in other Canadian provinces have indicated similar prevalence rates for these two pathogens (Lammerding et al. 1988; Arsenault et al. 2007). Agunos and coauthors reported the prevalence of Campylobacter isolated from retail chicken across Canada that ranged from 36% in the Maritimes to 42% in British Columbia (Agunos et al. 2014). In another three-year survey, Lammerding and colleagues identified that chicken carcasses collected across Canada were contaminated with Campylobacter (C. jejuni, C. coli and C. lari) at a rate of 38.2%.C.jejuni was the leading species with C. coli and C. lari being the second and third less frequent isolates, respectively (Lammerding et al. 1988). A more recent Canadian study tested chicken meats from retailers nationwide and detected Campylobacter in 48 out of 204 samples, yielding a contamination rate of 23.4% (Narvaez-Bravo et al. 2017). Arsenault and colleagues studied the prevalence of Campylobacter in broiler chickens before and after slaughtering in Quebec. They noted that the prevalence rate of Campylobacter on chicken carcasses was 35.8%, which was similar to our results. Furthermore, Arsenault and colleagues discovered that if more than 20% of birds had Campylobacter-positive cecal cultures or had jejunum contents (fecal matter) during slaughter, significantly higher rates 53 (P< 0.05) of contaminated chicken carcasses were observed following slaughter(Arsenault et al. 2007). In addition, a UK-based study found that the concentration of Campylobacter in the chicken intestinal contents at the time of slaughter can be as high as 107CFUg-1 (Newell 2002). Subsequently, these findings highlight the importance to implement pre-harvest control measures to reduce the contamination of this microbe in the final product. A closer look at the actions in New Zealand could shed some light on the nature and efficacy of the implemented control measures to reduce the prevalence and burden of campylobacteriosis in the country. It had been identified that fresh chicken meat was responsible for the majority of people being infected by Campylobacter at a really high incidence rate of 353.8/100,000 people between 2004 and 2006 (Sears et al. 2011). In 2008, the drastic removal of all fresh chicken from the retail market in New Zealand resulted in a 54% reduction in the annual rate of human infection to 161.5/100,000. Instead of fresh chicken meat, people were advised to consume frozen chicken meat which has been proven to have a significantly lower (up to >300 fold) Campylobacter contamination (Solow et al. 2003; Rosenquist et al. 2006). The freezing of chicken to prevent fresh chicken consumption appears to be an effective way to reduce illness but its implementation heavily depends on consumer opinion as it might not be popular to many consumers and might also pose certain challenges to the producers (Baker et al. 2006). Reducing contamination in poultry abattoirs by carcass washing and application of certain automated processing methods aiming to reduce contamination have also been validated to be challenging. Washing chicken carcasses can reduce Campylobacter concentrations by 0.5 to 1.5 log10 54 CFU/ml at the same time Campylobacter can be isolated in the poultry processing plants at rates up to 8 log10 CFU/mL in the rinse solution (Keener et al. 2004). In another Canadian study conducted in Saint Hyacinth, Quebec, the researchers discovered that broiler flocks could be colonized with multiple biotypes (Lior’s scheme) of C. jejuni and C. coli. They also discovered that ~20% of the macrorestriction profiles of the chicken isolates were the same as those identified in clinical isolates, indicating that chicken is a source of human infection and a public health hazard(Nadeau et al.2002). During a pilot experiment Schouls and coauthors discovered that a number of different subtypes could be isolated from an individual bird (up to 3 different C. jejuni biotypes) and potentially carry multiple Campylobacter species (Schouls et al. 2003). In this study, C. jejuni and C. coli were isolated simultaneously from 3 different chicken samples (3 lots of thighs) that were purchased from different locations. This emphasizes the need to develop isolation protocols that will target more than a single colony of Campylobacter and to identify different and emerging species and strains. Sample collection in our current study was based on the availability of chicken products on the local market which will reflect the dietary habits of the local population to some degree. The differences among the prevalence of Campylobacter on different chicken parts were not statistically significant (P> 0.05). Other studies have shown that there is a significant association with the presence of skin on chicken product and a higher Campylobacter load. Luber and coauthors detected Campylobacter on the surface of 87% of chicken samples that had the skin attached in contrast to only 20% of the deeper skinless and deboned tissue. Furthermore, the cell counts on positive samples of these two groups 55 differed by ~2 log CFU with the skinless samples carrying a lower Campylobacter load (Luber et al. 2007). In another study, Berrang and others noted a much higher difference between the samples with and without skin with Campylobacter concentrations from 50% to 90% higher in the samples with skin (Berrang et al. 2001). Chantarapanont and colleagues used confocal scanning laser microscopy to detect C. jejuni cells that had previously been transformed with the Pcgfp plasmid (green fluorescent protein or GFP-Campylobacter) and stained with 5-cyano-2,3-ditolyl tetrazolium chloride. After rinsing the inoculated chicken samples, researchers were able to visualize live Campylobacter cells via the red fluorescence located in skin crevices, entrapped in feather follicles as well as in the surface water layer. These findings highlight the role of the poultry skin to provide a suitable niche to enhance the survival of Campylobacter along the poultry processing line by preventing bacterial detachment from washing or contact with disinfectants (Chantarapanont et al. 2003). It also highlights the importance to implement strategies to minimize chicken skin cross-contamination during the evisceration stage where poultry skin can become contaminated by the intestinal contents (Hansson et al. 2015). In the current study, 46 samples were pre-packaged and 160were bulk samples (without packaging). The contamination rate of the prepackaged samples at 43.5% was significantly higher than that of the bulk samples at 31.8%, which is consistent with other studies (Lee et al. 1998). Packaging has been linked with higher survival rates of C. jejuni due to the microaerophilic atmospheric requirements of the pathogen as well as the preservation of moisture (Lee et al. 1998). In addition, several studies have proved that Campylobacter can survive for several weeks in moist and cool (4oC) environments 56 (Svedhem et al. 1981; Griffiths et al. 1990; Lu et al. 2011). Harrison and colleagues indicated that over 90% of supermarket pre-packaged chicken samples contained trapped moisture, which is believed to aid in the survival of Campylobacter (Harrison et al. 2001). The various production and preparation methods for different meat products have an impact on Campylobacter survival. Several studies have identified higher Campylobacter counts on whole chicken carcasses and chicken offal than in processed chicken parts, such as livers, gizzards, and hearts (Harrison et al. 2001; Stella et al. 2017; Baumgartner et al. 1995; Whyte et al. 2006). As the chicken meat moves along the processing line Campylobacter is challenged by several different environmental stresses, such as oxidative stress and drying (Stella et al. 2017). In the current study, we focused on the detection of Campylobacter on fresh chicken meat and offal and were only able to collect a small number of fresh chicken livers and gizzards since these types were commonly only available as frozen. All the chicken livers and almost half of the gizzards that we collected carried C. coli with only one gizzard carrying C. jejuni. In a study conducted in Tulsa, Oklahoma, the researchers detected a high prevalence of Campylobacter in chicken offal (67%) from which C. jejuni and C. coli accounted for 34% and 33% respectively. These isolates were tested for aerotolerance and the results indicated that 47% of them were aerotolerant (viable after a 12-h aerobic incubation period at 42oC) and 24% were hyper-aerotolerant (viable after a 24-h aerobic incubation). Interestingly, C. coli offal isolates demonstrated a greater aerotolerance (80%) compared to C. jejuni offal isolates (6%). These results could elucidate the strategy of aerotolerance in Campylobacter 57 spp. and its improved survival in the food chain (Karki et al. 2018; Noormohamed et al. 2012). In conclusion, the current study provides a comprehensive understanding of Campylobacter contamination on the retail level in metro-Vancouver. Our results showed that all different poultry products could pose an equal health risk to the public and also that prepackaged chicken could play a more significant role than bulk chicken meat in the development of human illness. Furthermore, we identified spring as the season showing the highest prevalence of the bacterium and thus, more consideration is needed in regards of the handling and cooking of raw chicken meat during that time period. Since poultry is considered as the major vehicle of human campylobacteriosis, further research is needed to find the most efficient control measures to adopt so as to reduce the prevalence of this pathogen in the province of British Columbia. 58 Chapter 3: Antimicrobial resistance profiles of C. jejuni and C. coli chicken isolates 3.1 Introduction Antimicrobial resistance testing aids in identifying the appropriate therapeutic regime and offering the surveillance and monitoring of resistance. Fluoroquinolones and macrolides are usually the first-line therapy for campylobacteriosis. The emergence of antimicrobial resistance to first-line therapy drugs emphasizes the importance of antimicrobial susceptibility testing for surveillance and to compare susceptibility to new and existing antimicrobial agents (Ge et al. 2013; EUCAST 2003). For some antimicrobials, resistance develops almost instantaneously whereas for some others it can take more than a decade before resistant strains appear (Schmieder et al. 2012). According to Gould and coauthors, the main factor influencing how rapidly resistance appears is the exposure dynamics of the resistome and this includes the levels of antibiotics and the time of exposure (Gould et al. 2002). The European Centers for Disease Control (ECDC) has established a clear correlation between antibiotic use and the development of resistance in European countries. For example, Greek physicians prescribe twice as many antibiotics per capita than Swedish doctors where resistance levels in Greece are much higher (Gullberg et al. 2011; ECDC 2011a, 2011b). The mechanisms conferring Campylobacter resistance to antimicrobials have been more extensively described in the introduction (section 1.9). 59 3.1.1 The minimum inhibitory concentration The minimum inhibitory concentration (MIC) of an antimicrobial agent is defined as the lowest concentration (mg/L) to which there is no visible growth of bacteria and is defined in vitro within a specific time period (EUCAST 2003). Therefore, MIC is a method that is used to standardize the susceptibility testing of microorganisms. It can also be used as a reference method from which to judge the performance of other susceptibility testing methods (Andrews 2002). In the dilution tests, Campylobacter can be tested for its ability to produce visible growth on a series of agar plates (agar dilution) or in broth (broth dilution) containing two-fold dilutions of the antimicrobial agents. The MIC is the concentration below the lowest concentration with visible bacterial growth (Figure 3.1). When there is bacterial growth in all the tested concentrations, the highest concentration is regarded as the MIC value. Both the broth and agar dilution methods are quantitative and although there is a good correlation between the two, the agar dilution method is more laborious and time consuming (Elshikh et al. 2016). In the broth dilution method, vessels containing identical volumes of broth with known and increasing concentrations of antibiotics are inoculated with a culture of known bacterial numbers. The testing is usually conducted in 96 well microdilution plates (≤ 300μL/well). The results can be interpreted by the naked eye or a plate reader (by measuring the optical density) and characterized as reproducible to within one well (two-fold dilution) divergence of the real end point (EUCAST 2003). 60 3.1.2 Breakpoints Breakpoints (or susceptibility test interpretive criteria by FDA) were established by the European Centre for Antimicrobial Susceptibility Testing (EUCAST) in the 1970s and are used in the characterization of bacterial strains in phenotypic antimicrobial susceptibility tests. The main parameters used in the determination of breakpoints include dosages, pharmacokinetics, resistance mechanisms, MIC distributions, inhibition zone diameters and more recently pharmacodynamics and epidemiological cutoff values (ECOFFs) (Mouton et al. 2012). Breakpoints are used to characterize microorganisms as susceptible (S), intermediate (I), or resistant (R) depending on the quantitative antimicrobial susceptibility as indicated by the MIC value. The definition of susceptible by EUCAST is that “A microorganism is defined as susceptible by a level of antimicrobial activity associated with a high likelihood of therapeutic success. A microorganism is categorized as susceptible by applying the appropriate breakpoint in a defined phenotypic system” (Kahlmeter 2003; Kahlmeter et al. 2006).In comparison, resistance is defined as a high likelihood of therapeutic failure (Mouton et al. 2012). 3.2 Materials and methods 3.2.1 Antimicrobial solutions Each Campylobacter isolate was tested to determine its antimicrobial resistance (AMR) against six clinically important antimicrobial classes, including aminoglycoside (gentamicin [GEN]), lincosamides (clindamycin [CLI]), macrolides (erythromycin [ERY] and azithromycin[AZI]), quinolones (ciprofloxacin [CIP] and nalidixic acid [NAL]), 61 tetracyclines (tetracycline [TET] and doxycycline [DOX]), and phenicols (chloramphenicol [CHL] and florfenicol [FLO]). The listed antimicrobial agents (above) were purchased from Sigma-Aldrich (Oakville, ON, Canada) and weighed out and dissolved to provide stock and working solutions according to the manufacturer’s guidelines. Table 3.1 shows the dilutions and solvents used to produce stock solutions for each of the ten antimicrobial agents. The concentrations were adjusted according to the purity of each antimicrobial compound as described by the manufacturer. Mueller-Hinton broth was used as the diluent to produce the working solutions for all the antimicrobials. Aliquoted stock solutions were stored at -20oC until use. The 96-well micro-titration plates were purchased from VWR (Mississauga, ON, Canada). A calibrated analytical balance was used to weigh the antimicrobial agents and the solutions were prepared with allowance for the potency of powder using the following formula: 𝑊𝑒𝑖𝑔ℎ𝑡𝑜𝑓𝑝𝑜𝑤𝑑𝑒𝑟 (𝑚𝑔) =𝑉𝑜𝑙𝑢𝑚𝑒𝑜𝑓𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛(𝑚𝐿)𝑥𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝑔𝐿)𝑃𝑜𝑡𝑒𝑛𝑐𝑦𝑜𝑓𝑝𝑜𝑤𝑑𝑒𝑟 (𝑚𝑔𝑔) According to the European Committee of Antimicrobial Testing (EUCAST) sterilization of the antimicrobial solutions is not necessary. If solutions are sterilized, membrane filtration could be applied and samples before and after sterilization should be compared to ensure that adsorption has not occurred (EUCAST 2003). 62 Table 3.1 Dilutions of the antimicrobial agents to produce the stock solutions Antimicrobial agent Purity Dilution Solvent nalidixic acid ≥98% 50 mg/mL chloroform ciprofloxacin ≥98% 25 mg/mL hydrochloric acid clindamycin 87% 20 mg/mL water azithromycin 95% 20 mg/mL ethanol (100%) gentamicin 66% 10 mg/mL water chloramphenicol ≥98% 50 mg/mL ethanol (100%) tetracycline 95% 10 mg/mL water erythromycin 85% 50 mg/mL ethanol (100%) doxycycline ≥98% 50 mg/mL ethanol (100%) florfenicol ≥98% 50 mg/mL dimethyl sulfoxide A range of concentrations of the antimicrobial agents were loaded into the 96 well plates on the basis of a previous publication by Ma and others (Ma et al. 2017). For all the antimicrobial agents that were tested, 12 different concentrations, each diluted two-fold, were used with the highest at64 μg/mL and the lowest at 0.03 μg/mL. Briefly, 200μL of the antimicrobial at 64μg/mL was loaded into all the wells in column 1and the remainder wells on the plate (column 2-12) were loaded with 100 μL MH (Mueller-Hinto) broth. Two-fold dilutions were created by pipetting 100 μL from each well of column 1 to each well on column 2 and re-suspended 20 times. This 2-fold serial dilution was repeated for each consecutive column through to column 12 where the remaining 100 μL of the last dilution was discarded (European Society of Clinical Microbiology and Infectious Diseases 2019). 3.2.2 Campylobacter inoculations Campylobacter chicken isolates that had previously been stored at -80oC were streaked on Mueller-Hinton blood agar plates and incubated at 42oC under microaerobic 63 conditions for 48 h. Bacterial colonies were harvested from the MHA (Mueller-Hinton agar) plates and suspended in Mueller-Hinton broth and incubated at 37oC under microaerobic conditions for 16-18 h in a rotatory manner (i.e. 175 rpm). Each culture was adjusted to OD600nm = ~0.3 (1 × 109 CFU/mL) and then further diluted in MH broth to a final concentration of 2 x 108 CFU/mL, a suspension equivalent of 0.5 McFarland standard (Lubbers et al. 2018). According to the Clinical and Laboratory Standards Institute (CLSI) guidelines, McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions to be within a given concentration range (CLSI 2015). The 96-well plates that were previously loaded with the range of different antimicrobials were inoculated with 100 μL of 2x108 CFU/mL of each Campylobacter isolate culture resulting in a bacterial cell suspension of 1x108 CFU/mL in each well and were incubated microaerobically at 42oC for 48 h. Each isolate was tested in duplicate and two biological replicates (testing on different days) were performed. In total, four MIC values were collected for each antimicrobial agent for each Campylobacter isolate. A total of 58Campylobacter isolates were tested for their resistance to 10 different antimicrobial agents belonging to 6 different classes. C. jejuni ATCC 33560 from the laboratory inventory stock was used as the quality control (QC) strain to validate each test and was included in every AMR assay on Campylobacter chicken isolates. The ranges of the acceptable MICs of the QC strain are included in Table 3.2. 64 Table 3.2 Antimicrobial test ranges, MIC QC range, and breakpoints used for antimicrobial susceptibility testing* MIC breakpoint (μg/mL) Antimicrobial class Antimicrobial agent MIC QC range (μg/mL) Test range (μg/mL) S I R Aminoglycoside gentamicin 0.5-4 0.03-64 ≤2 4 ≥8 Lincosamides clindamycin 0.12-0.5 0.03-64 ≤2 4 ≥8 Quinolones ciprofloxacin 0.06-0.5 0.03-64 ≤1 2 ≥4 nalidixic acid 4-16 0.03-64 ≤16 32 ≥64 Macrolides erythromycin 1-8 0.03-64 ≤8 16 ≥32 azithromycin 0.03-012 0.03-64 ≤2 4 ≥8 Tetracyclines tetracycline 0.25-1 0.03-64 ≤4 8 ≥16 doxycycline 0.5-2 0.03-64 ≤2 4 ≥8 chloramphenicol 1-4 0.03-64 ≤8 16 ≥32 florfenicol 0.5-2 0.03-64 ≤4 8 ≥16 *QC, quality control; S, susceptible isolate; I, intermediate isolate; R, resistant isolate. 3.3 Results 3.3.1 Antimicrobial resistance (AMR) profiles of Campylobacter isolates The MICs of the QC strain C. jejuni ATCC 33560 were within the reference quality control range during all experiments (Table 3.2). Table 3.2 also includes the MIC breakpoints used in our study as previously described in another study (Ma et al. 2017). In the current study, the MICs of Campylobacter isolates were recorded macroscopically (visually) as shown in Figure 3.1. Based on the selected breakpoints, the majority of C. jejuni (37/45) and C. coli (9/13) isolates (82.2% and 69.2% respectively) were resistant to at least one antimicrobial class. Subsequently, a smaller number of isolates were pan- 65 susceptible (susceptible to all of the antimicrobial agents) at a rate of 17.8% and 30.8% for C. jejuni (n=8) and C. coli (n=4),respectively (Table 3.3). Figure 3.2 shows the analytical data of antimicrobial resistance rates of two Campylobacter species to all the antimicrobial agents tested in the current study. Figure 3.1 Visual MICs of a C. jejuni strain in Mueller-Hinton broth. Antimicrobials tested in rows A – H: ciprofloxacin, erythromycin, clindamycin, tetracycline, chloramphenicol, gentamicin, florfenicol, and nalidixic acid, respectively. The wells showing the MIC values are circled in red. 66 Figure 3.2 Antimicrobial resistance of C. jejuni and C. coli isolates. ERY, erythromycin; AZI, azithromycin; TET, tetracycline; DOX, doxycycline; GEN, gentamicin; CLI, clindamycin; CIP, ciprofloxacin; NAL, nalidixic acid; CHL, chloramphenicol; FLO, florfenicol. The majority (80%) of C. jejuni isolates (27/45) were resistant to at least one quinolone (ciprofloxacin and/or nalidixic acid) with 4.4% (n=2) being resistant only to ciprofloxacin and 26.7% (n=12) being resistant exclusively to nalidixic acid. The remainder 42.3% (n=13) were resistant to both of the tested quinolones (CIP-NAL). No C. jejuni isolate was resistant to tetracycline or doxycycline alone. However, 1 isolate (2.2%) was 0 02022.20 051.1600 00 038.4 38.40 038.469.20 001020304050607080ERY AZI TET DOX GEN CLI CIP NAL CHL FLOResistance %Antimicrobial agentC. jejuniC. coli 67 resistant to both tetracycline and doxycycline (TET-DOX) and another C. jejuni isolate (2.2%) showed resistance to one quinolone agent (nalidixic acid) and one tetracycline agent (doxycycline) (NAL-DOX). Finally, 17.8% (n=8) of the isolated C. jejuni were resistant to 4 antimicrobial agents belonging in two different antimicrobial classes, namely quinolones and tetracyclines (CIP-NAL-TET-DOX) (Table 3.3). Multidrug resistance defined as phenotypic non-susceptibility to 3 or more classes of antimicrobials was not observed in any of the C. jejuni isolates. In comparison, 30.8% (n=4)C. coli isolates exhibited resistance to nalidixic acid alone. No C. coli isolate exhibited resistance to the other quinolone (ciprofloxacin) or both quinolones (CIP-NAL). Similarly, no C. coli isolates were resistant to any of the tetracyclines alone or paired. However, 38.5% (n=5) C. coli isolates were simultaneously resistant to 4 antimicrobials including ciprofloxacin, nalidixic acid, tetracycline and doxycycline from the tetracycline and quinolone class and at a relatively higher rate than observed for C. jejuni (17.8%) (Table 3.3). Similarly, multidrug resistance (≥ 3 classes) was not observed in any of the C. coli isolates. 68 Table 3.3 Antimicrobial resistance profiles of C. jejuni and C. coli isolates No. of antimicrobial classes No. of C. j. isolates % of C. j. isolates No. of C.c. isolates % of C. c. isolates CIP 1 2 4.4 0 0 NAL 1 12 26.7 4 30.8 CIP-NAL 1 13 28.9 0 0 TET-DOX 1 1 2.2 0 0 NAL-DOX 2 1 2.2 0 0 CIP-NAL-TET-DOX 2 8 17.8 5 38.5 Multidrug resistance 3 0 0 0 0 Susceptible 0 8 17.8 4 30.8 Total - 45 - 13 - 3.4 Discussion The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) is a surveillance system coordinated by the Public Health Agency of Canada, aiming to monitor the use of antimicrobial agents in humans as well as the antimicrobial resistance in selected representative bacterial species (Salmonella, E. coli and Campylobacter) that are important to human and animal health. The agri-food component of this program performs surveillance on the antimicrobial use in animal husbandry along with the prevalence of the aforementioned bacterial species as well as their AMR patterns. CIPARS is performed on farms, in abattoirs and at retail level. Since 2005, CIPARS has performed the surveillance of Campylobacter at the abattoir level for beef cattle and since 69 2010 for broiler chickens. The surveillance of retail chicken meat has been going on for much longer since2003. However, there is currently no available data from surveillance measures on Campylobacter isolated from humans (Ge et al. 2013; PHAC 2018). According to the CIPARS report in 2011, Campylobacter isolated from Canadian retail chicken exhibited the highest rates of resistance to the tetracyclines (45.8%), followed by much lower rates for quinolones and macrolides (6.9% and 2.9%) (PHAC 2012). In the current study, C. jejuni had a resistance rate of 22.2%to tetracyclines when including resistance profiles that contained TET and/or DOX. On the other hand, resistance rates to tetracyclines were slightly higher for C. coli with 38.5% of the isolates exhibiting resistance to both tetracycline and doxycycline. In a recent two-year survey, Varga and coauthors studied the levels of tetracycline resistance of Campylobacter that had been isolated from small poultry flocks in Ontario. Researchers found that up to 77% of C. jejuni isolated from chickens were resistant to tetracycline and C. jejuni had a higher resistance to tetracycline than C. coli (36%) (Varga et al. 2019). In contrast, we detected that tetracycline resistance was much lower (22.2%) in C. jejuni than C. coli (38.5%).Similar to our findings, Varga and others noted that multidrug resistant profiles were uncommon in Campylobacter isolates from chicken. The difference in the resistance rates of isolated Campylobacter spp. to tetracyclines in the current study to those from Ontario could possibly be explained by different antimicrobial practices employed by veterinarians and farmers to treat animal diseases across the country. According to the National Antimicrobial Resistance Monitoring System’s (NARMS) report for the period between 2007 and 2011, C. jejuni isolates were most 70 commonly resistant to tetracycline, followed by ciprofloxacin and nalidixic acid (FDA/CDC/USDA 2013). During this 5-year period, ciprofloxacin resistance decreased in C. jejuni from 25.8% in 2007 to 23.5% in 2011 while resistance in C. coli increased from 28.6% in 2007 to 35.8% in 2011. Macrolide resistance was observed at very low levels (1.7% for C. jejuni and 2.7% for C. coli). Gentamicin resistance of C. coli increased from 0 in 2007 to 12.2% in 2011. In the CIPARS report for the period between 2005 and 2010 it was noted that ciprofloxacin resistance in retail chicken increased in British Columbia by 13% (from 3.6% to 17.1%) (Agunos et al. 2013). In the current study, the rates appeared even higher, with 51.1% of C. jejuni and 38.6% of C. coli isolates showing resistance to ciprofloxacin often in combination with other drug resistance patterns. The Canadian poultry industry has developed a policy to eliminate the prophylactic use of fluoroquinolones (listed by Health Canada and World Health Organization as Category I drug of importance to human health) that have been effective since May 2014, practically banning the use of ciprofloxacin in poultry production. However, according to the Public Health Agency of Canada, resistance of Campylobacter spp. to this drug has continued to increase from 17.1% to 48.3% in BC over the past five years (PHAC 2006; WHO 2019). Furthermore, surveillance data from the US indicated that despite the 2005 ban on the fluoroquinolone use in chickens, the resistance rates have not dropped since. It has been suggested that once resistance to fluoroquinolones has been generated it is successfully maintained within Campylobacter population due to having no fitness cost in the absence of quinolones (Agunos et al. 2013). With approximately 141 million poultry chicks and eggs being imported to Canada from the US annually, this might be an additional source of 71 antimicrobial resistant Campylobacter to the Canadian poultry industry (Agunos et al. 2013) The surveillance data by CIPARS has also shown that the resistance of Campylobacter from retail chicken in Canada was less frequently resistant to macrolides (2.9%) and lincosamides (clindamycin 1.4%). Furthermore, no resistance was observed to gentamicin and florfenicol (PHAC 2012). Similarly, Campylobacter isolates in this study showed noresistance to macrolides, lincosamides and phenicols. Data from the European Union (2011) shows a similar trend of antimicrobial resistance in Campylobacter isolates. The most commonly reported resistance to antimicrobials were nalidixic acid (47.8%), followed by ciprofloxacin (44.4%), ampicillin (35.5%) and tetracycline (30.5%). Resistance to macrolides and aminoglycoside were also reported to be less frequent (3.5% and 0.4% respectively) in Europe (Ge et al. 2013). The newest report from the European Food Safety Authority (EFSA) shows an overall increase in ciprofloxacin and tetracycline resistance at 57.5% and 45.5%, respectively. Some European countries exhibit extremely high rates in ciprofloxacin resistance, including Portugal (96.5%), Lithuania (91.5%), Spain (88.6%), Estonia (84.0%) and Cyprus (80.0%). Similarly, over 80% of Campylobacter isolates from Cyprus, Malta, Portugal and Spain were resistant to tetracycline (EFSA 2017). Several reports suggest that macrolides and specifically azithromycin should be used as an alternative drug of choice to treat campylobacteriosis due to the increased levels of resistance to fluoroquinolones (Tribble 2017; Ge et al. 2013; Agunos et al. 2013). Azithromycin may also be preferred to treat campylobacteriosis as erythromycin may have 72 a slight increase in risk of cardiac arrest, particularly when given to individuals on medication that inhibits the enzyme cytochrome P450 3A4 (Ray et al. 2004). Resistance to both azithromycin and erythromycin remains low in the United States with less than 2% of C. jejuni and 9% of C. coli being reported to be resistant in 2012(CDC 2019). Tribble and colleagues noted that a 1-g dose of azithromycin for 3 days had a 96% efficacy in curing patients with Traveler’s diarrhea in Thailand compared to the 3 day regime using 500 mg of levofloxacin (Tribble et al. 2007). These findings were also supported by another study conducted by Vukelic and colleagues that compared a daily dose of azithromycin to a 5-day regimen with erythromycin. Their results showed that treatment with azithromycin was significantly superior to that with erythromycin (Vukelic et al. 2009). In the current study, all Campylobacter isolates were susceptible to azithromycin at MICs ≤ 0.06 μg/mL. Varga and colleagues discovered low levels of multidrug resistant Campylobacter when they screened small flocks of poultry for antimicrobial resistance in Ontario. They noticed a higher resistance of C. coli to azithromycin than that in C. jejuni. This could be explained by the fact that macrolide resistance is supported by the substitutions in the 23S rRNA gene and specifically at A2075G and less frequently at A2074C/G. In C. jejuni, this mutation has been associated with decreased colonization capability in chicken that was not similarly associated with C. coli (Bolinger et al. 2017). Furthermore, Varga and coauthors observed a very low frequency of antimicrobial resistance of the isolated Campylobacter to aminoglycoside (gentamicin) and ketolides (telithromycin). Similarly, we observed that all Campylobacter isolates were susceptible to gentamicin and phenicols (chloramphenicol, florfenicol). 73 Taken together, our current study revealed the absence of multi-drug resistance profiles in Campylobacter isolated from retail chicken in metro-Vancouver. We discovered a high level of resistance of two thermotolerant Campylobacter species to nalidixic acid at 60% for C. jejuni and 69.2% for C. coli, followed by resistance to ciprofloxacin at 51.1% and 38.4% for C. jejuni and C. coli respectively and lastly by tetracycline and doxycycline (20% and 22% for C. jejuni) and 38.4% for both tetracycline agents for C. coli. The absence of resistance of the isolated strains to all the other antimicrobial agents that were tested, namely erythromycin, azithromycin, clindamycin, gentamicin, chlorphenicol and florfenicol possibly indicate the moderate use of these agents in animal husbandry and in the treatment of human illness in Canada. Therefore, we could suggest that macrolides, aminoglycoside and phenicols could be used as the drugs of choice to treat human campylobacteriosis in British Columbia. 74 Chapter 4: The multilocus sequence typing of C. jejuni and C. coli isolated from chicken and associations with antimicrobial resistance and Canadian clinical isolates. 4.1 Introduction One of the strengths of MLST lies in the revelation of all the variations in a single locus and that it only requires the sequencing of a small (of 450-500 bp) fragment of the housekeeping genes to give unambiguous information about the genetic relationship of bacteria (Enright et al. 1999). This small size is ideal for accurate sequencing and provides sufficient nucleotide variation in each locus to be highly discriminatory, where a single nucleotide change will be assigned as a different allele number (Dingle et al. 2005). Changes in the housekeeping genes are a slow process. As a result of their slow evolution, they exhibit a minimal variation on the targeted sequences, making MLST an ideal method for global epidemiology. For Campylobacter, the following seven loci are used in the MLST scheme for both C. jejuni and C. coli: aspA (aspartase A), glnA (glutamine synthetase), gltA (citrate synthase), glyA (serine hydroxymethyltransferase), pgm (phosphoglucomutase), tkt (transketolase), and uncA (ATP synthase α subunit); their protein products are shown in parentheses (Maiden et al. 1998; Dingle et al. 2001; https://Pubmlst.Org/Campylobacter) The number of alleles per locus can range from 1 to 430and allele numbers can be assigned on the PubMLST website from the uploading of sequences (Thépault et al. 2017). The 7 allele numbers (one for each of the seven loci) provide the allelic profile to which a 75 single number or sequence type (ST) is assigned (https://Pubmlst.Org/Campylobacter/). STs that share four or more allele numbers are more closely related and belong to the same clonal complex (CC). The fact that all the alleles of the seven loci and their variations determine the allelic profile of an isolate makes the results unambiguous as the possibility for a ST to be occurring by chance is ~ 1/10,000 (Enright et al. 1999). MLST is readily transferable between laboratories by uploading the isolates along with the corresponding allelic profiles and ST’s onto the PubMLST online database, making sequence data from various sources and locations sharable. It is well established that C. jejuni exhibits a high diversity in regards of virulence and pathogenicity traits, i.e. adherence, invasion, toxicity and molecular mimicry (Coote et al. 2007; Zheng et al. 2006; AbuOun et al. 2005; Datta et al. 2003). When a closer look at the virulence of a strain is of interest, the porA and flaA gene loci can be included in the MLST scheme. The porA gene encodes the major outer membrane protein (MOMP) of C. jejuni and C. coli and it exhibits a high genetic diversity and has been linked with the cases of human campylobacteriosis (Cody et al. 2009). In addition, the expression of the flaA gene has been clearly associated with the ability of Campylobacter to adhere and penetrate host cells. Subsequently, typing of both porA and flaA genes provides valuable information about the epidemiology of the strains. 76 4.2 Materials and methods 4.2.1 Campylobacter isolates and preparation of DNA template Based on their AMR profiles (Chapter 3), a selection of C. jejuni (n=15) and C. coli (n = 9) retail chicken isolates (Chapter 2) were plated onto MH agar plates from the glycerol stock (-80oC) and incubated at 42oC for at least 24 h under microaerophilic conditions. Campylobacter DNA was extracted using a PrestoTM Mini gDNA Bacteria Kit (FroggaBio, Toronto, ON, Canada) according to the manufacturer’s instructions. DNA concentration was determined by NanoDrop (Thermo Scientific, United States). The isolates selected for MLST were those exhibiting different MICs (S, I, R) to the range of antimicrobial agents tested. For C. jejuni, 4 pan-susceptible and 11 isolates showing antimicrobial resistance were selected out of the 45 AMR profiles. More specifically, of the resistant C. jejuni profiles, five isolates were resistant to nalidixic acid, one isolate was resistant to nalidixic acid and ciprofloxacin, one isolate was resistant to tetracycline and doxycycline and three isolates were resistant to ciprofloxacin, nalidixic acid, tetracycline and doxycycline. For C. coli (total of 13 AMR profiles), we included one pan-susceptible isolate, one isolate resistant to ciprofloxacin, two isolates resistant to ciprofloxacin and nalidixic acid and five isolates resistant to ciprofloxacin, nalidixic acid, tetracycline and doxycycline. 4.2.2 Amplification and nucleotide sequence determination Table 4.1 and Table 4.2 show the primers used for the amplification of the seven loci for C. jejuni and C. coli. The primer sets for both Campylobacter species have been 77 previously described (Miller et al. 2005; https://pubmlst.org/campylobacter/info/primers.shtml). Table 4.1 Oligonucleotide primers for MLST of C. jejuni amplification Locus Primer sequence forward (5’-3’) Primer sequence reverse (5’-3) Amplicon size aspA GAGAGAAAAGCWGAAGAATTTAAAGAT TTTTTTCATTWGCRSTAATACCATC 676 glnA TGATAGGMACTTGGCAYCATATYAC ARRCTCATATGMACATGCATACCA 751 gltA GARTGGCTTGCKGAAAAYAARCTTT TATAAACCCTATGYCCAAAGCCCAT 706 glyA ATTCAGGTTCTCAAGCTAATCAAGG GCTAAATCYGCATCTTTKCCRCTAAA 716 pgm CATTGCGTGTDGTTTTAGATGTVGC AATTTTCHGTBCCAGAATAGCGAAA 720 tkt GCAAAYTCAGGMCAYCCAGGTGC TTTTAATHAVHTCTTCRCCCAAAGGT 730 uncA GWCAAGGDGTTATYTGTATWTATGTTGC TTTAADAVYTCAACCATTCTTTGTCC 700 Table 4.2 Oligonucleotide primers for MLST of C. coli amplification Locus Primer sequence forward (5’-3’) Primer sequence reverse (5’-3) Amplicon size (bp) aspA CAACTTCAAGATGCAGTACC ATCTGCTAAAGTATGCATTGC 573 glnA TTCATGGATGGCAACCTATTG GCTTTGGCATAAAAGTTGCAG 594 gltA GATGTAGTGCATCTTTTACTC AAGCGCTCCAATACCTGCTG 508 glyA TCAAGGCGTTTATGCTGCAC CCATCACTTACAAGCTTATAC 606 pgm TTATAAGGTAGCTCCGACTG GTTCCGAATAGCGAAATAACAC 652 tkt AGGCTTGTGTTTTCAGGCGG TGACTTCCTTCAAGCTCTCC 580 uncA AAGCACAGTGGCTCAAGTTG CTACTTGCCTCATCCAATCAC 598 Two PCR assays were used, one for each species with some modifications. For C. jejuni, each amplification reaction mixture consisted of 0.5 μL of DNA template, 2 μL 78 of 10x PCR buffer, 50 μM MgCl2, 0.1 μM of each deoxynucleoside triphosphate (dNTPs), 0.1 μM of each gltA, glyA and pgm primers, 0.2 μM of each uncA and glnA primers,0.3 μM of each aspA and tkt primers and 0.5 U of Platinum™ Taq DNA Polymerase (Thermo Fisher Scientific, Toronto, Canada). The remainder made up of nuclease-free water to a final volume of 20μL. For C. coli, we used the same reaction as described for C. jejuni with a single primer concentration of 0.1 μM for all seven sets of primers. For the DNA amplification, we used the Veriti 96-well Thermal Cycler (Applied Biosystems, Foster City, CA) where the reaction included a denaturation step at 94°C for 2 min, followed by 35 cycles of amplification (denaturation at 95°C for 0.5 min, annealing at 57°C for 0.5 min and extension at 72°C for 1 min), ending with a final extension at 72°C for 10 min. The PCR products were stained with 1.5% (v/v) SYBR safe™ and gel electrophoresis was performed using 2% agarose and visualized using a Bio-Rad ChemiDoc MP gel imaging system (Bio-Rad, Hercules, CA). Once a single and discrete band of each expected size was observed each PCR product was aliquoted in 2 separate PCR tubes containing 5 μL of the PCR product, 2 μL of either forward or reverse primers (only one primer was added in each PCR tube) and 7 μL of nuclease free water to a total volume of 14μL. The samples were sequenced at the Sequencing and Bioinformatics Consortium (SBC), Vancouver, BC. 4.3 Results 4.3.1 Detection of housekeeping genes in C. jejuni and C. coli isolates MLST was used to detect and assign allele numbers to the seven housekeeping genes in Campylobacter. The two thermotolerant Campylobacter species produced seven 79 amplicons each. For C. jejuni the amplicon sizes were as expected at676 bp, 751 bp, 706 bp, 716 bp, 720 bp, 730 bp and 700 bp for aspA, glnA, gltA, glyA, pgm, tkt, and uncA, respectively. Similarly, for C. coli the DNA fragments had the expected molecular weights of 573 bp, 594 bp, 508 bp, 606 bp, 652 bp, 580 bp and 598 bp for the same loci, respectively (Figure 4.1). Figure 4.1 Gel electrophoresis of PCR products for MLST scheme A) C. jejuni, B) C. coli. Lane M: molecular weight marker, 100-bp DNA ladder; Lane 1-7: aspA, uncA, gltA, glyA, pgm, tkt, and glnA, respectively. 80 4.3.2 Assignment of allele numbers, STs and clonal complexes The sequences were uploaded as chromat files on the BioNumerics v. 7.6 (Applied Maths, Belgium) software and were queried through the software against the Campylobacter PubMLST database. The allelic profile (7 allele numbers) of each isolate was automatically identified along with the previously assigned ST and CC number. The novel STs that did not exist in the PubMLST database were submitted individually in both the sequence and the profile databases and the novel ST numbers were assigned overnight. Overall, the MLST typing of 15 C. jejuni isolates identified nine STs belonging to 7 clonal complexes, namely ST-939 (CC-353), ST-42 (CC-42), ST-267 (CC-283), ST-982 (CC-21), ST-10115 (CC-21), ST-354 (CC-354), ST-353 (CC-353), ST-48 (CC-48) and one singleton ST-1911 (Table 4.3). C. jejuni ST-10115was a new ST assigned through the MLST database. In the current study, the most common C. jejuni belonged to CC-283 at 26.7% (n= 4), CC-21 at 20% (n= 3) and CC-42 at 13.3% (n= 2). For C. coli, MLST was able to separate the isolates into five different STs (ST-1082, ST-829, ST-10116, ST-10117 and ST-10119) all of which belong to CC-828 clonal complex except ST-10116 which is a singleton (Table 4.4). The latter 3 ST’s (ST-10116, ST-10117 and ST-10119) were novel and were submitted to the PubMLST database and were assigned a ST number. 81 Table 4.3 Allele numbers, sequence types and clonal complexes of C. jejuni isolates* MLST profile Clonal complex ST No. of isolates aspA glnA gltA glyA pgm tkt uncA CC-353 939 1 7 2 5 2 156 3 6 CC-42 42 2 1 2 3 4 5 9 3 CC-283 267 4 4 7 40 4 42 51 1 CC-21 982 1 2 1 2 3 2 1 5 CC-21 10115 3 2 1 2 10 2 3 5 CC-354 354 1 8 10 2 2 11 12 6 CC-353 353 1 7 17 5 2 10 3 6 CC-48 48 1 2 4 1 2 7 1 5 Singleton 1911 1 7 84 5 10 119 178 26 *Novel STs are in boldface. Table 4.4 Allele numbers, sequence types and clonal complexes of C. coli isolates MLST profile Clonal complex ST No. of isolates aspA glnA gltA glyA pgm tkt uncA CC-828 1082 1 33 39 30 82 211 85 17 CC-828 10117 1 33 39 30 82 113 35 84 CC-828 10119 2 33 39 30 82 113 43 84 CC-828 829 3 33 39 30 82 113 43 17 Singleton 10116 2 6 4 30 82 113 1 5 *Novel STs are in boldface. 82 We also used BioNumerics v. 7.6 to create minimum spanning trees to illustrate the relationship between chicken and human isolates for the two thermotolerant Campylobacter species. Furthermore, we created phylogenetic trees to investigate the genetic relationship among C. jejuni and C. coli chicken isolates in relationship with their AMR patterns. The minimum spanning tree created from C. jejuni strains isolated from chicken and human stools showed that all of the identified STs belonged to clonal complexes that also contain clinical isolates (Figure 4.2). The high genetic diversity of C. jejuni shown in Figure 4.2 is consistent with other previous studies (Stone et al. 2013; Manning et al. 2003;Dingle et al. 2001). Similarly, for C. coli, the majority of the identified STs clustered together showing a strong relationship (sharing 4-6 alleles)between them as well as with human clinical isolates with the novel singleton ST-10116 appearing a little more distant with only 3 shared alleles with ST-829, ST-10116 and ST-10119 (Figure 4.3). 83 Figure 4.2 Minimum spanning tree established on the 9 C. jejuni STs identified in this study and human isolate STs from Canada collected from the MLST database. Each circle represents an ST. The size of the circle is positively correlated to the isolate numbers 84 with the same ST. The branch styles represent the numbers of identical alleles between connected STs (black thick solid line, six alleles; black thin solid line, five alleles; gray thick solid line, four alleles; dashed line, three alleles; and dotted line, two alleles or fewer). The color inside the circle stands for the clonal complex. Chicken and human isolates are shown with yellow and black frames, respectively. Figure 4.3 Minimum spanning tree established on 5 C. coli STs isolated from retail chicken in this study and compared to human C. coli isolates in Canada acquired from the MLST database. Each circle represents an ST. The size of the circle is proportional to the isolate numbers with the 85 same ST. The branch styles represent the numbers of identical alleles between connected STs (black thick solid line, six alleles; black thin solid line, five alleles; gray thick solid line, four alleles; dashed line, three alleles; and dotted line, two alleles or fewer). The color inside the circle stands for clonal complex. Chicken isolates are shown with a thick yellow border of the circle. 4.3.3 Relationship between MLST and antimicrobial resistance The phylogenetic trees in Figures 9a and 9b depict the connection between sequence types and their corresponding antimicrobial resistance profiles from this study (Chapter 3). Generally, isolates with the same ST have the same AMR profile. Although some C. jejuni isolates were attributed to the identical clonal complex, they appeared slightly distant in the phylogenetic tree and presented different antimicrobial resistance profiles. For example, ST-10115 and ST-982 were assigned to the ST-21 complex. The three ST-10115isolates are close to each other in the phylogenetic tree, and both were resistant to ciprofloxacin, nalidixic acid, tetracycline, and doxycycline, whereas ST-982 appears in a neighboring branch and exhibited resistance only to tetracyclines (tetracycline and doxycycline). 86 Figure 4.4 Phylogenetic tree of C. jejuni isolates with the relative clonal complex and resistance profile. Figure 4.5 Phylogenetic tree of C.coli isolates with the relative clonal complex and resistance profile. 87 The majority (75%) of the C. coli ST’s isolated from retail chicken belonged to the ST-828 clonal complex as shown in Table 4.4 and Figure 4.3. Figure 4.5 shows the link between the identified STs and their antimicrobial resistance. The distance among different C. coli isolates appears to be equally proportional in the two figures. For example, the minimum spanning tree shows a closer relationship between ST-829 and ST-10119 and then ST-10117. The other STs, ST-1082 and ST-10116 appear to share fewer alleles with the rest of the C. coli isolates. The phylogenetic tree shows an identical relationship between the isolates as in the minimum spanning tree. However, it becomes clear that the same ST might present different AMR profiles. For example, ST-829 was isolated three times at different times and locations; one isolate (ST-829) was resistant to four antimicrobial agents belonging to two different classes, namely ciprofloxacin, nalidixic acid, tetracycline and doxycycline. The other two ST-829 isolates were resistant to both of the antimicrobial agents of only one class, the quinolones. 4.4 Discussion Clonal complex ST-21 is one of the largest clonal complexes identified to date, constituting about one third of all of the submitted C. jejuni isolates with more than one hundred different sequence types(Best et al. 2004; https://Pubmlst.Org/Campylobacter/). This clonal complex is frequently associated with the cases of human disease and has been found in a wide range of food chain sources such as chickens, cattle, ruminants, and environmental waters (Dearlove et al. 2016). This makes it difficult for MLST to attribute a single host reservoir to a specific clonal complex. It has been suggested that specific Campylobacter lineages show host “generalism” and that it is possible for host-restricted sub-lineages to exist within these clonal 88 complexes (Gripp et al. 2011; Sheppard et al. 2014; Dearlove et al. 2016). Gripp and colleagues used a subset of closely related C. jejuni strains from the commonly isolated ST-21 (sequence type) from different hosts and isolation sources to investigate the diversity of this common ST that was isolated from different sources. The sources included chickens, pigs, cattle, wild birds, monkeys, pet animals, human patients, and food sources. Phenotypic tests examining metabolic variation, protein expression patterns and cell interaction demonstrated a high phenotypic diversity within the ST-21 sequence types, which did not correlate with a specific isolation source (Gripp et al. 2011).Whole genome sequencing was applied to shed some light into the genomic variation of the strains. Again, the diversity was remarkable, primarily due to recombination and acquisition of phage-related genes. Gripp and others also stated that no genomic feature was discovered that could be associated with a specific host (Gripp et al. 2011). In the current study, we identified four C. jejuni isolates belonging to 2 different sequence types that were assigned to ST-21 complex. Three of them were resistant to quinolones (CIP-NAL) and tetracyclines (TET-DOX) and one was only resistant to tetracyclines (TET-DOX). Interestingly, we observed that when a strain from ST-21 complex was resistant to one antimicrobial agent in one class it was also to the other agent in the same class. In other words, we did not find a strain belonging to ST-21 complex being only resistant to either ciprofloxacin or nalidixic acid alone. While investigating the genetic similarity of ciprofloxacin resistant Campylobacter in central Europe, Kovac and coauthors noted that a mutation in the quinolone resistance-determining region of the gyrA gene was conferring ciprofloxacin resistance to ST-21 and ST-353 complex (Kovač et al. 2015). These clonal complexes were the most commonly identified in 89 the study with ST-21 including 61 isolates and ST-353 including 26 isolates respectively, from a total of 178 STs. The authors noted that both ST-21 and ST-353 were highly represented by chicken and human isolates and no cattle isolates were assigned to ST-353 complex. Interestingly, in Africa, the same clonal complex contained strains isolated from broiler flocks that were completely susceptible to ciprofloxacin (Kinana et al. 2006).Another study conducted in north China also showed that ST-353 was among the most popular clonal complexes to be identified and only included isolates from broiler flocks, ranking second only after ST-21 complex (Zhang et al. 2010). Zhang and colleagues discovered that isolates belonging to the ST-353 complex were resistant to ciprofloxacin, levofloxacin and nalidixic acid (quinolones). In another study, certain clonal complexes were shown to be highly host associated and that this was greater than the geographical signal (Méric et al. 2014). In a later study from the same group, ST-353 and ST-354 were identified to only include chicken and human isolates (Sheppard et al. 2010). In the current study, we identified two C. jejuni isolates belonging to the ST-353 complex and one belonging to and ST-354 complex. The AMR testing revealed that all three were pan-susceptible. The ST-283 complex has also been strongly associated with both human and chicken isolates (Olkkola et al. 2016; De Haan et al. 2010; Shin et al. 2013). It has also been suggested that it is a possible generalist complex and whole genome sequencing has revealed common ancestry between ST-283 and ST-45 (Sheppard et al. 2014; Dearlove et al. 2016). In previous studies, it has been reported that ST-283 showed no resistance to macrolides but was moderately resistant to ciprofloxacin. ST-283 complex was more commonly isolated during the summer months (Cody et al. 2012). Similarly, we discovered that all four of our C. jejuni isolates 90 belonging to the ST-283 complex were susceptible to both macrolides (erythromycin and azithromycin) and ciprofloxacin. However, they were also resistant to another drug from the quinolone class, namely nalidixic acid. Lastly, 3 out of 4of our isolates that belonged to ST-283 complex were isolated during the warmer months and one in winter. The application of MLST also identified a C. jejuni isolate (ST-42) belonging to the ST-42 complex. Other studies have shown that this complex is commonly associated with isolates from chickens, humans and cattle. In fact, Kwan and colleagues discovered that ST-42 was the third most common C. jejuni sequence type to be isolated from cattle at a frequency of 20% after ST-61 complex (24.2%) and ST-21 complex (23.6%) (Kwan et al. 2008). The phylogenetic tree in Figure 4.4 clearly demonstrates that ST-42 is weakly related to other C. jejuni isolates included in this study. Nevertheless, the minimum spanning tree in Figure 4.2 shows a very close genetic relationship between the ST-42 that we isolated from chicken and the human isolates from the database. In a UK based study, the majority of strains (75%) belonging to ST-42 complex were resistant to ciprofloxacin but susceptible to erythromycin (Cody et al. 2012). Similarly, in the current study ST-42 was resistant to quinolones and susceptible to macrolides. One of the C. jejuni isolates that we identified (ST-48) was assigned to the ST-48 complex. Dingle and coauthors investigated the epidemiology of Campylobacter samples from various sources and noted that the ST-48 complex was one of the most commonly encountered clonal complexes, along with ST-21, ST-45, ST-206, ST-61 and ST-257 (Dingle et al. 2005). ST-48 was reported to contain C. jejuni strains predominantly isolated from human clinical samples, cattle and sand from beaches, but also from chickens and ruminants in lower frequencies. The 91 phylogenetic tree of C. jejuni (Figure 4.4) reveals the closer linkage between ST-48 and ST-21, which is also a generalist clonal complex. Dearlove and coauthors studied C. jejuni and C. coli clonal complexes that were commonly associated with human disease and identified a dominant C. coli complex, namely ST-828 that did not exhibit any host association and appeared to be one of the generalist complexes as well (Dearlove et al. 2016). A comparison between other similar studies reveals that ST-828 has been identified in many countries and from various sources (humans, sheep, cattle, chickens, pigs, water) as the predominant C. coli complex (Litrup et al. 2007; Thakur et al. 2006; Dearlove et al. 2016). It was reported that in New Zealand, Scotland and Switzerland, 54%, 94% and 71% of the isolated C. coli population belonged to ST-828 clonal complex, emphasizing its global distribution (Nohra et al. 2016).In a study conducted in Oklahoma, Noormohamed and colleagues also discovered that ST-828 was the most common clonal complex containing C. coli isolated from chicken. In the same study, ST-828 clonal complex was most commonly associated with human isolates (Noormohamed et al. 2014). The majority (78%) of our C. coli isolates belonged to only one clonal complex (ST-828). This could be due to the more conservative nature of C. coli genome while C. jejuni genome being more variable (Wassenaar et al. 1998; Dingle et al. 2005). Among the 7 C. coli that were assigned to ST-828 complex, we observed a variety of different antimicrobial resistance patterns. One strain was pan-susceptible whereas the other three showed resistance to 4 different antimicrobials in two different classes (ciprofloxacin, nalidixic acid, tetracycline and doxycycline). In conclusion, MLST subtyping of C. jejuni and C. coli previously isolated from retail chicken revealed a variety of STs belonging to commonly identified clonal complexes (ST-21, 92 ST-42, ST-48, ST-283, ST-343, ST-353 and ST-828). The majority of the clonal complexes that we discovered are believed to show generalism as they have been isolated from various sources (chickens, humans, cattle, pigs, ruminants, environmental water and soil) in different geographical regions. Furthermore, we also identified two clonal complexes that show high host association and more specifically, they have been linked with poultry and human clinical samples only. These findings highlight the possible linkage between the consumption or mishandling of chicken meat contaminated with these STs of C. jejuni and C. coli and some cases of human campylobacteriosis in Canada. 93 Chapter 5: Conclusion 5.1 Study outcomes In this thesis project, we investigated the prevalence of C. jejuni and C. coli on the retail level in metro Vancouver. We discovered a total Campylobacter prevalence of 34% with C. jejuni and C. coli accounting for 80% and 20% of the isolates, respectively, without any other Campylobacter species being identified. Furthermore, the comparison between the prevalence rates of Campylobacter in different chicken samples showed no significant difference. However, we discovered a higher Campylobacter prevalence rate (P < 0.05) in the packaged than that in the bulk chicken meat. Furthermore, we identified both thermotolerant Campylobacter species being simultaneously present on three different chicken samples. A clear seasonal trend of Campylobacter prevalence was observed. In the current study, the total prevalence of Campylobacter was highest in spring (91%), followed by summer (54%), autumn (28.6%) and winter (10.2%). A high level of resistance of Campylobacter chicken isolates to quinolones was discovered. More specifically 60% of C. jejuni and 69.2% of C. coli were resistant to nalidixic acid, followed by resistance to ciprofloxacin at 51.1% and 38.4% for C. jejuni and C. coli, respectively. In addition, tetracycline and doxycycline resistance were at 20% and 22% respectively for C. jejuni and 38.4% for both tetracycline agents for C. coli. Contrary to our initial hypothesis, we did not discover any multi-drug resistant Campylobacter belonging to either species. Nevertheless, 17.8% of C. jejuni and 38.5% of C. coli were resistant to ciprofloxacin, nalidixic acid, tetracycline and doxycycline at the same time. Lastly, we identified that C. jejuni and C. coli were pan-susceptible at 17.8% and 30.8%, respectively. 94 MLST subtyping revealed a high genetic diversity among 24 Campylobacter strains derived from the same host (chicken). A total of 9 C. jejuni STs (ST-21, ST-42, ST-48, ST-267, ST-343, ST-353, ST-982, ST-939 and ST-10115) were identified belonging to six commonly identified clonal complexes (ST-21, ST-42, ST-48, ST-283, ST-343 and ST-353), with ST-10115 being a novel ST and ST-1911 being a singleton. Additionally, 9 C. coli isolates were sequenced and we discovered five STs (ST-829, ST-1082, ST-10116, ST-10117, ST-10119) from which four were identified to belong to only one clonal complex namely, ST-828, with three STs being novel (ST-10116, ST-10117, ST-10119) and ST-10119 also being a singleton. These results provide a comprehensive understanding of the prevalence, antimicrobial resistance and genetic diversity of Campylobacter in poultry products in metro Vancouver. As poultry products are the major reservoir of this microbe and are the most commonly implicated foods with human campylobacteriosis, further mitigation strategies should be developed to control the prevalence of this pathogen in the agri-foods systems. As Campylobacter poses a major risk to human infections at the retail and home level, more research and education of the public is needed to implement strategies to control Campylobacter contamination. 5.2 Future directions Currently, whole genome sequencing (WGS) is considered as the most informative and discriminative typing method of bacterial pathogens (Llarena, Taboada, and Rossi 2017). WGS may be a useful alternative to the prediction of antibiotic resistance in Campylobacter as it allows for comprehensive phylogenetic analysis of numerous traits associated with antibiotic resistance (Fiedoruk et al. 2019; Whitehouse et al. 2018; S. Zhao et al. 2016). While studying the 95 molecular determinants associated with antibiotic resistance, Fabre and colleagues identified two new gene variants putatively associated with gentamicin resistance, a gene likely associated with lincosamide resistance as well as two resistance enzymes similar to those identified in Staphylococcus aureus (Fabre et al. 2018). These findings suggest that WGS is a powerful tool to reveal the full genetic elements that confer resistance in Campylobacter. Finally, WGS or whole genome MLST (wgMLST) would provide sequence data that would allow genome comparisons and recognition of genetically related isolates within the STs aiding in rapid detection of potential contamination sources and identifying outbreaks (Kovanen et al. 2014). 96 Bibliography Aarestrup, F. M., Engberg, J. 2001. “Antimicrobial resistance of thermophilic Campylobacter.” Veterinary Research 32: 311–21. Aarestrup, F. M., Nielsen, E. 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Thesis/Dissertation
eng
Food Science
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Prevalence and characterization of Campylobacter isolated from chicken products in metro Vancouver
Text